Photochemical internalization for delivery of molecules into the cytosol

ABSTRACT

The present invention provides a method for introducing a molecule into the cytosol of a cell in which the cell is contacted with a photosentistising agent, the cell is irradiated with light of a wavelength effective to activate the photosentisitising agent and, substantially at the same time or after the irradiation, the cell is contacted with the molecule to be introduced, particularly for use in cancer treatment, gene therapy and vaccination.

The present invention relates to an improved method for introducingmolecules into the cytosol of cells using a photosensitising agent andirradiation of the cells with light of a wavelength effective toactivate the photosensitising agent.

The majority of molecules do not readily penetrate cell membranes.Methods for introducing molecules into the cytosol of living cells areuseful tools for manipulating and studying biological processes. Amongthe most commonly used methods today are microinjection, red blood cellghost-mediated fusion and liposome fusion, osmotic lysis of pinosomes,scrape loading, electroporation, calcium phosphate and virus-mediatedtransfection. These techniques are useful for investigating cells inculture, although in many cases they may be impractical, time consuming,inefficient or they may induce significant cell death. Thus suchtechniques are not optimal for use in biological or medical research, orin therapies where it is required that cells should remain viable and/orfunctional.

It is well known that porphyrins and many other photosensitizingcompounds may induce cytotoxic effects on cells and tissues. Theseeffects are based upon the fact that upon exposure to light thephotosensitizing compound may become toxic or may release toxicsubstances such as singlet oxygen or other oxidising radicals which aredamaging to cellular material or biomolecules, including the membranesof cells and cell structures, and such cellular or membrane damage mayeventually kill the cells. These effects have been utilised in thetreatment of various abnormalities or disorders, including especiallyneoplastic diseases. The treatment is named photodynamic therapy (PDT)and involves the administration of photosensitizing(photochemotherapeutic) agents to the affected area of the body,followed by exposure to activating light in order to activate thephotosensitizing agents and convert them into cytotoxic form, wherebythe affected cells are killed or their proliferative potentialdiminished. Photosensitizing agents are known which will localisepreferentially or selectively to the desired target site e.g. to atumour or other lesion.

A range of photosensitizing agents are known, including notably thepsoralens, the porphyrins, the chlorins and the phthalocyanins. Suchdrugs become toxic when exposed to light.

Photosensitizing drugs may exert their effects by a variety ofmechanisms, directly or indirectly. Thus for example, certainphotosensitisers become directly toxic when activated by light, whereasothers act to generate toxic species, e.g. oxidising agents such assinglet oxygen or other oxygen-derived free radicals, which areextremely destructive to cellular material and biomolecules such aslipids, proteins and nucleic acids.

Porphyrin photosensitisers act indirectly by generation of toxic oxygenspecies, and are regarded as particularly favourable candidates for PDT.Porphyrins are naturally occurring precursors in the synthesis of heme.In particular, heme is produced when iron (Fe³⁺) is incorporated inprotoporphyrin IX (PpIX) by the action of the enzyme ferrochelatase.PpIX is an extremely potent photosensitizer, whereas heme has nophotosensitizing effect. A variety of porphyrin-based orporphyrin-related photosensitisers are known in the art and described inthe literature.

The cytotoxic effect of most sensitizers used in PDT is mediated mainlythrough the formation of singlet oxygen formed upon exposure of thephotosensitizers to light. This reactive intermediate has a very shortlifetime in cells (<0.04 μs) Thus, the primary cytotoxic effect of PDTis executed during light exposure and very close to the sites offormation of ¹O₂. ¹O₂ reacts with and oxidizes proteins (histidine,tryptophan, methionine, cysteine, tyrosine), DNA (guanine), unsaturatedfatty acids and cholesterol. One of the advantages of PDT is thattissues unexposed to light may be left unaffected ie. that a selectivePDT effect may be obtained. There is extensive documentation regardinguse of PDT to destroy unwanted cell populations, for example neoplasticcells. The patent literature describes a number of photodynamiccompounds, alone or conjugated with targeting agents, e.g.immunoglobulins directed to neoplastic cell receptor determinants,making the complex more cell specific. Certain photochemical compounds,such as hematoporphyrin derivatives, have furthermore an inherentability to localise in malignant cells. Such methods and compounds, aredescribed in the Norwegian patent No. 173319 and in Norwegian patentapplications Nos. 90 0731, 176 645, 176 947, 180 742, 176 786, 301 981,30 0499 and 89 1491.

In WO93/14142 a drug delivery system is described which comprises ananti-cancer agent and a photoactivatable agent (ie. a photosensitizer)attached to copolymeric carriers. Upon administration this complexenters the cell interior by pinocytosis or phagocytosis and locatesinside the endosomes and lysosomes. In the lysosomes, the bond betweenthe anti-neoplastic agent and the polymer is hydrolysed and the formercan diffuse passively through the lysosome membrane into the cytosol.The utility of this method is thus limited to small molecular compoundswhich are able to diffuse across the lysosome membranes. After allowinga time lag for diffusion, a light source of appropriate wavelength andenergy is applied to activate the photo-activatable compound. Thecombined effect of the anti-cancer agent and photoactivatable agentdestroy the cell. Such PDT methods as described above are thus directedto the destruction of cell structures leading to cell death.

WO 96/07432 and WO 00/54802 on the other hand, are concerned withmethods which use the photodynamic effect as a mechanism for introducingotherwise membrane-impermeable molecules into the cytosol of a cell in amanner which does not necessarily result in widespread cell destructionor cell death. In this method, the molecule to be internalised and aphotosensitising compound are applied simultaneously or in sequence tothe cells, upon which the photosensitizing compound and the molecule areendocytosed or in other ways translocated into endosomes, lysosomes orother intracellular membrane restricted compartments.

The molecule to be translocated into intracellular compartments of thecells and the photosensitising compound are applied to the cellstogether or sequentially and are taken up by the cell together into thesame intracellular compartments (i.e. are co-translocated). The moleculeto be internalised within the cell is then released by exposure of thecells to light of suitable wavelengths to activate the photosensitisingcompound which in turn leads to the disruption of the intracellularcompartment membranes and the subsequent release of the molecule, whichis located in the same compartment as the photosentizing agent, into thecytosol. This method was termed “photochemical internalisation” or PCI.Thus, in these methods the final step of exposing the cells to lightresults in the molecule in question being released from the sameintracellular compartment as the photosensitizing agent and becomingpresent in the cytosol.

It was believed that in order for this method to be effective it wasessential that both the photosensitising compound and the molecule to bereleased into the cytosol were present in the same intracellularcompartments when irradiation was performed.

It has now surprisingly been found that molecules can be introduced intothe cytosol of cells by similar PCI methods but where the exposure ofthe cells to light is not necessarily the final step and the methods arenot dependent on the molecule and the photosensitizing agent beinglocated in the same intracellular compartments at the time of lightexposure. In such methods the photosensitising agent may be contactedwith the cells and activated by irradiation before the molecule to beinternalised and thus delivered to the cytosol is brought into contactwith the cells. Thus, despite the fact that the molecule to beinternalised and the photosensitising agent are not necessarilylocalised in the same intracellular compartments at the time of lightexposure, the molecule still enters the cell and is delivered to thecytosol. These results are extremely surprising and such methods displaysignificant advantages over the methods where light irradiation is thefinal step.

At its most general therefore, the present invention provides a methodfor introducing a molecule into the cytosol of a cell, said methodcomprising contacting said cell with a photosensitising agent,contacting said cell with the molecule to be introduced and irradiatingsaid cell with light of a wavelength effective to activate thephotosensitising agent, wherein said irradiation is performed prior tothe cellular uptake of said molecule into an intracellular compartmentcontaining said photosensitising agent, preferably prior to cellularuptake of said molecule into any intracellular compartment.

Thus in one alternative, said irradiation can be performed after thecellular uptake of the molecule into an intracellular compartment,providing said molecule to be internalised and the photosensitisingagent are not localised in the same intracellular compartments at thetime of light exposure. In a preferred embodiment however irradiation isperformed prior to cellular uptake of the molecule to be internalised.

“Internalisation” as used herein, refers to the cytosolic delivery ofmolecules. In the present case “internalisation” thus includes the stepof release of molecules from intracellular/membrane bound compartmentsinto the cytosol of the cells.

As used herein, “cellular uptake” or “translocation” refers to one ofthe steps of internalisation in which molecules external to the cellmembrane are taken into the cell such that they are found interior tothe outerlying cell membrane, e.g. by endocytosis or other appropriateuptake mechanisms, for example into or associated with an intracellularmembrane-restricted compartments, for example the endoplasmic reticulum,Golgi body, lysosomes, endosomes etc.

In particular, viewed from a preferred aspect the present inventionprovides a method for introducing a molecule into the cytosol of a cell,said method comprising contacting said cell with a photosensitisingagent, irradiating said cell with light of a wavelength effective toactivate the photosensitising agent and, substantially at the same timeor at a time after the irradiation, contacting said cell with themolecule to be introduced.

Preferably the cells are contacted with the molecules to be introducedor internalised (referred to hereinafter as the transfer molecules) at atime point after irradiation has taken place, or in other words,photochemical treatment of the cells, by contacting them with aphotosensitising agent and then irradiation, is effected before themolecules are added to the cells. In this embodiment the molecules to beintroduced into the cytosol can be brought into contact with the cellswhich have been subjected to photochemical treatment at any time pointafter the treatment has occurred providing the transfer molecules arestill able to be taken up into the cells. The time window in which themolecules may be brought into contact with the cells and still be takenup may depend on a variety of factors such as for example the cell type,the particular molecule in question, the particular photosensitisingagent used, and the duration of the light treatment. This time windowcan if necessary be determined for a particular set of conditions.However, preferably the molecule to be transferred to the cytosol isexposed to the cells relatively soon after photochemical treatment, forexample within 24 hours after photochemical treatment and morepreferably within the first 10 hours after photochemical treatment e.g.within the first 5 hours or more preferably the first hour. For examplein vitro or ex vivo the transfer molecule may be administered for acertain time period, e.g. 30 minutes to 24 hours, preferably 1 to 2hours, commencing administration immediately after or shortly afterirradiation, e.g. if the end of irradiation is considered as the startpoint, the transfer molecule may be applied at 0 minutes to 24 hours,e.g. 0 to 4 hours.

It has been observed that even if the transfer molecule is contactedwith the cell a considerable time after irradiation, internalizationinto the cell is still possible. Thus, for example, the transfermolecule may be applied more than an hour after irradiation, e.g. morethan 2, 4, 8, 10 or even 12 hours after irradiation.

Thus, in a preferred embodiment said cell is contacted with saidtransfer molecule 0 to 4 hours after irradiation for a period of 1 to 2or 3 hours or longer, e.g. at least 0.5 to 3 hours. The time at whichthe transfer molecule is administered will vary depending on whether themethods are being carried out in vitro or in vivo. For in vitro methodsthe transfer molecules can generally be brought into contact with allthe target cells simultaneously, e.g. if the cells are growing in an invitro culture and thus it is relatively easy to bring the molecules incontact with the cells at an appropriate time point. In vivo however,the step of contacting the target cells with the transfer molecules isclearly more complicated and will depend on the mode of administrationand the location of the target cells. For example, where the transfermolecule can be administered directly to the target cells, e.g. by localinjection, then the transfer molecule will come into contact with thetarget cells (or at least a proportion of them) relatively quickly, e.g.in a matter of minutes or hours after administration. If on the otherhand the transfer molecules are administered by intravenous injectionfor a distant target then these molecules may take a lot longer to comeinto contact with the target cells. For example they may take 24 to 96hours after administration to reach the target cells. This “journeytime” will have to be taken into account in deciding the appropriatetime for which to administer the transfer molecules relative to theadministration of the photosensitizing agent and the time ofirradiation.

In an alternative embodiment of the invention rather than the transfermolecule being brought into contact with the cells after irradiation hastaken place it can be brought into contact with the cells substantiallyat the same time as the irradiation. “Substantially at the same time” asused herein includes exactly at the same time i.e. simultaneously, butalso includes the addition of the molecule to the cells shortly beforeirradiation, for example up to one or two hours before irradiation,providing that the cellular uptake of the transfer molecule has notoccurred at the time of irradiation and may still occur, albeit afterirradiation or providing that if cellular uptake of the transfermolecule has occurred then the transfer molecule and thephotosensitizing agent are not localised to the same intracellularcompartments at the time of light exposure.

As mentioned above, the precise timing of the addition of the transfermolecule and photosensitizing agent and timing of irradiation to achievethe above described effects need to take into account various factorsincluding the cells to be treated, agents and molecules in use and theenvironment of the cells, particularly with regard to whether an invitro or in vivo system is in issue. Taking these considerations intoaccount appropriate timings may readily be determined.

As a general principle appropriate conditions are determined such thatthe irradiation step should take place either prior to the cellularuptake of the transfer molecule (assuming that the photosensitizingagent itself has been take up into intracellular compartments) or afterthe cellular uptake of the transfer molecule provided that the transfermolecule and the photosensitizing agent are not located in the sameintracellular compartments at the time of light exposure. In this latterscenario, clearly the transfer molecule will come into contact with thecells at a time point before irradiation takes place. This provides oneof the preferred embodiments of the present,invention.

Previously disclosed methods of photochemical internalisation whereinthe transfer molecule and the photosensitising agent were added to thecells prior to irradiation depended on the molecules in question beinglocated in the same intracellular compartments prior to light exposureso that lysis of these compartments by the light activation of thephotosensitising agent resulted in the release of both the molecule andthe photosensitising agent into the cytosol. A schematic drawing showingthis is shown in FIG. 7.

In the present methods clearly the photosensitising agent and thetransfer molecule to be introduced into the cytosol are not in the sameintracellular compartments at the time of light exposure since thetransfer molecule is only added to the cells shortly before or after thecells are exposed to light.

The mechanism of action of the present methods is as yet unknown andindeed the fact that this method works at all is surprising. Whilst notwishing to be bound by theory, the reason for these surprising findingsmay be that fusion of photochemically damaged vesicles with newly formedendocytotic vesicles takes place which is then followed by the releaseof newly endocytosed molecules into the cytosol. A schematic diagramillustrating this is shown in FIG. 7. Alternatively, photochemicaldamage to lysosomal enzymes or vesicles containing lysosomal enzymes,such as late endosomes, may reduce the rate of intracellular degradationof the molecules to be internalized. This may be due to reducedtransport to vesicles containing lysosomal enzymes or transport toendocytic vesicles containing lower hydrolytic activity. In this waythese molecules will have more time to escape the endocyticcompartmentalization than when the lysosomal degradation pathway isactive. A further alternative explanation could be that thephotochemical treatment of the cells leads to minor damage to the plasmamembrane of the cells leading to increased penetration of macromoleculesthrough the cell membrane. However experiments carried out (see Example7) suggest that this is probably not the reason.

The present invention thus relates to methods for transporting ortransfecting any molecules into the cytosol of living cells either invitro (i.e. in culture) or in vivo, after which the molecules shall beavailable in the cytosol.

Such methods can be used not only to transfer molecules (or parts orfragments thereof) into the interior of a cell but also, in certaincircumstances, to present or express them on the cell surface. Thus,following transport and release of a transfer molecule into the cellcytosol according to the methods of the present invention, if thecell(s) in question are specialised cells, such as for example antigenpresenting cells, the molecule or fragment, may be transported to thesurface of the cell where it may be presented on the outside of the cellie. on the cell surface. Such methods have particular utility in thefield of vaccination, where vaccine components ie. antigens orimmunogens, may be introduced into a cell for presentation on thesurface of that cell, in order to induce, facilitate or augment animmune response. Further details as to the utility of being able toexpress molecules on the cell surface are described in WO 00/54802.

The transfer molecules which can be introduced into the cytosol of cellsusing the methods of the present invention include molecules which donot readily penetrate cell membranes. Additionally, the presentinvention can increase the cytosol delivery and activity of moleculeswhich are only partly able to penetrate the membrane of the cell or themembranes of intracellular vesicles. Transfer molecules may be organiccompounds, proteins or fragments of proteins such as for examplepeptides, antibodies or antigens or fragments thereof. Another class oftransfer molecules for use according to the invention are cytotoxicdrugs such as protein toxins or cytotoxic organic compounds, e.g.bleomycin. Still another class of appropriate transfer molecules arenucleic acids.

Nucleic acids may be used in the form of genes encoding for exampletherapeutic proteins, antisense RNA molecules, ribozymes, RNA aptamersor triplex forming oligonucleotides. Alternatively the nucleic acids maybe employed in the form of non-encoding molecules such as for examplesynthetic DNA or RNA antisense molecules, ribozymes, aptamers, triplexforming oligonucleotides, peptide nucleic acids (PNAs), transcriptionfactor “decoy” DNA or chimeric oligonucleotides for repair of specificmutations in the patient. Where appropriate the nucleic acid moleculesmay be in the form of whole genes or nucleic acid fragments optionallyincorporated into a vector molecule or entity e.g. a plasmid vector or aviral particle or bacteriophage. The latter form has particularapplicability when the transfer molecule is to be used in methods ofgene therapy.

The photosensitizing agent to be used according to the present inventionis conveniently any such agent which localises to intracellularcompartments, particularly endosomes or lysosomes. A range of suchphotosensitising agents are known in the art and are described in theliterature, including in WO96/07432. Mention may be made in this respectof di- and tetrasulfonated aluminium phthalocyanine (e.g. AlPcS_(2a)),sulfonated tetraphenylporphines (TPPS_(n)), nile blue, chlorin e₆derivatives, uroporphyrin I, phylloerythrin, hematoporphyrin andmethylene blue which have been shown to locate in endosomes andlysosomes of cells in culture. This is in most cases due to endocyticuptake of the photosensitizer. Thus, the photosensitizing agent ispreferably an agent which is taken up into the internal compartments oflysosomes or endosomes. However, other photosensitizing agents whichlocate to other intracellular compartments for example the endoplasmicreticulum or the Golgi apparatus may also be used. It is alsoconceivable that mechanisms may be at work where the effects of thephotochemical treatment are on other components of the cell (i.e.components other than membrane-restricted compartments). Thus, forexample one possibility may be that the photochemical treatment destroysmolecules important for intracellular transport or vesicle fusion. Suchmolecules may not necessarily be located in membrane-restrictedcompartments, but the photochemical damage of such molecules maynevertheless lead to photochemical internalisation of the transfermolecules, e.g. by a mechanism where photochemical effects on suchmolecules lead to reduced transport of the molecule to be internalized(i.e. the transfer molecule) to degradative vesicles such as lysosomes,so that the molecule to be internalized can escape to the cytosol beforebeing degraded. Examples of such molecules not necessarily located inmembrane restricted compartments are several molecules of themicrotubular transport system like dynein and components of dynactin;and for example rab5, rab7, N-ethylmaleimde sensitive factor (NSF),soluble NSF attachment protein (SNAP) and so on.

Classes of suitable photosensitising agents which may be mentioned thusinclude porphyrins, phthalocyanines, purpurins, chlorins,benzoporphyrins naphthalocyanines, cationic dyes, tetracyclines andlysomotropic weak bases or derivatives thereof (Berg et al., J.Photochemistry and Photobiology, 1997, 65, 403–409). Other suitablephotosensitising agents include texaphyrins, pheophorbides, porphycenes,bacteriochlorins, ketochlorins, hematoporphyrin derivatives, andderivatives thereof, endogenous photosensitizers induced by5-aminolevulinic acid and derivatives thereof, dimers or otherconjugates between photosensitizers.

Preferably the photosensitizer is in free form, ie. not conjugated toany other macromolecule. However the photosensitizer may alternativelybe associated with, attached to, or conjugated to, a carrier or othermolecule as described hereinafter, e.g. attached to a targeting antibodyor coupled to a carrier such as polylysine.

Preferred photosensitising agents include TPPS₄, TPPS_(2a), AlPcS_(2a)and other amphiphilic photosensitizers. In a preferred aspect, thepresent invention provides methods in which the photosensitizing agentswhich may be used are compounds being 5-aminolevulinic acid or esters of5-aminolevulinic acid or pharmaceutically acceptable salts thereof.

In such esters the 5-amino group may be substituted or unsubstituted,the latter case being the ALA esters.

More particularly, the ALA esters for use according to the invention areesters of 5-aminolevulinic acids with optionally substituted alkanols,ie. alkyl esters or substituted alkyl esters.

Conveniently, ALA esters which may be used are compounds of formula I,R₂ ²N—CH₂COCH₂—CH₂CO—OR¹  (I)(wherein R¹ may represent alkyl optionally substituted by hydroxy,alkoxy, acyloxy, alkoxycarbonyloxy, amino, aryl, oxo or fluoro groupsand optionally interrupted by oxygen, nitrogen, sulphur or phosphorusatoms; and R², each of which may be the same or different, represents ahydrogen atom or a group R¹) and salts thereof.

The substituted alkyl R¹ groups may be mono or poly-substituted. Thussuitable R¹ groups include for example unsubstituted alkyl, alkoxyalkyl,hydroxyalkoxyalkyl, polyhydroxyalkyl, hydroxy poly alkyleneoxyalkyl andthe like. The term “acyl” as used herein includes both carboxylate andcarbonate groups, thus, acyloxy substituted alkyl groups include forexample alkylcarbonyloxy alkyl. In such groups any alkylene moietiespreferably have carbon atom contents defined for alkyl groups below.Preferred aryl groups include phenyl and monocyclic 5–7 memberedheteroaromatics, especially phenyl and such groups may themselvesoptionally be substituted.

Representative substituted alkyl groups R¹ include alkoxymethyl,alkoxyethyl and alkoxypropyl groups or acyloxymethyl, acyloxyethyl andacyloxypropyl groups eg. pivaloyloxymethyl.

Preferred ALA esters for use as photosensitizing agents according to theinvention, include those wherein R¹ represents an unsubstituted alkylgroup and/or each R² represents a hydrogen atom.

As used herein, the term “alkyl” includes any long or short chain,straight-chained or branched aliphatic saturated or unsaturatedhydrocarbon group. The unsaturated alkyl groups may be mono- orpolyunsaturated and include both alkenyl and alkynyl groups. Such groupsmay contain up to 40 carbon atoms. However, alkyl groups containing upto 10 eg. 8, more preferably up to 6, and especially preferably up to 4carbon atoms are preferred.

Particular mention may be made of ALA-methylester, ALA-ethylester,ALA-propylester, ALA-hexylester, ALA-heptylester and ALA-octylester andsalts thereof, which represent preferred photosensitizing agents for useaccording to the invention.

Necessarily, the photosensitising agent is contacted with a cell priorto irradiation. However, unlike the transfer molecule, this agent shouldbe administered sufficiently prior to irradiation such that onirradiation said agent has been taken up into an intracellularcompartment. Thus conveniently said agent is applied 1 to 72 hours priorto irradiation, e.g. 4 to 48 e.g. 4 to 24 hours prior to irradiation.Again, as discussed above in connection with the step of bringing thetransfer molecule into contact with the cells, the timing ofadministration of the photosensitizing agent to achieve contact with thetarget cell in relation to the time point of irradiation will depend onthe time it will take for a photosensitizing agent to reach the targetcells and be taken up by them. This time may vary depending on whetherthe methods are being carried out in vitro or in vivo and on whether theadministration is direct to the target tissue or is at a distal site. Inall cases, it is important that the photosensitizing agent has beentaken up by the target cells before irradiation takes place. Said agentmay be maintained in contact with said cells immediately up toirradiation, e.g. for 1 or 4 to 72 hours, preferably 4 to 24 hours, e.g.12 to 20 hours, or may be removed from contact immediately prior toirradiation, e.g. for more than 5 minutes, e.g. for 10 minutes to 8hours, e.g. 1 hour to 4 hours in agent-free medium.

Optionally, one or other or both of the photosensitising agent and thetransfer molecule to be introduced into cells may be attached to orassociated with or conjugated to one or more carrier molecules,targetting molecules or vectors which can act to facilitate or increasethe uptake of the photosensitising agent or the transfer molecule or canact to target or deliver these entities to a particular cell type,tissue or intracellular compartment. Examples of carrier systems includepolylysine or other polycations, dextran sulphate, different cationiclipids, liposomes, reconstituted LDL-particles, sterically stabilisedliposomes or adenovirus particles. These carrier systems can generallyimprove the pharmacokinetics and increase the cellular uptake of thetransfer molecule and/or the photosensitizing agent and may also directthe transfer molecule and/or the photosensitizing agent to intracellularcompartments that are especially beneficial for obtaining photochemicalinternalisation, but they do not generally have the ability to targetthe transfer molecule and/or the photosensitizing agent to specificcells (e.g. cancer cells) or tissues. However, to achieve such specificor selective targeting the carrier molecules, the transfer moleculeand/or the photosensitizer may be associated or conjugated to specifictargetting molecules that will promote the specific cellular uptake ofthe transfer molecule into desired cells or issues. Such targettingmolecules may also direct the transfer molecule to intracellularcompartments that are especially beneficial for obtaining photochemicalinternalisation.

Many different targeting molecules can be employed, e.g. as described inCuriel, D. T. (1999), Ann. New York Acad. Sci. 886, 158–171; Bilbao, G.et al. (1998), in Gene Therapy of Cancer (Walden et al., eds., PlenumPress, New York), Peng K. W. and Russell S. J. (1999), Curr. Opin.Biotechnol. 10, 454–457, Wickham T. J. (2000), Gene Ther. 7, 110–114.

The carrier molecule and/or the targetting molecule may be associated,bound or conjugated to the transfer molecule, to the photosensitizingagent or both, and the same or different carrier or targeting moleculesmay be used. If for example adenovirus particles are used as carriersthen the transfer molecules may be incorporated within the adenovirusparticles. For example if the transfer molecule in question is a DNAmolecule encoding a protein or an RNA molecule, then the DNA isincorporated into the virus vector and after photochemicalinternalisation the DNA molecule will be present at the correctintracellular location so that expression of the encoded molecule canoccur.

Expression of such molecules can be controlled by designing the vectorby methods well known and documented in the art. For example, regulatoryelements such as for example tissue specific or regulatable promoterscan be used to obtain tissue or disease specific or regulatableexpression. For example the tissue specific promoter melanoma specifictyrosinase promoter may be used. Regulatable promoters such astetracylin-regulated promoters are well known. More examples of specificor regulated promoters that can be employed in the present invention canbe found in Hart, I. R., 1996, Semin. Oncol. 23, 154–158; Hallahan, D.E. et al., 1995, Nature Med. 1, 786–791; Luna, M. C. et al. 2000, CancerRes. 60, 1637–1644; Miller, N. and Whelan, J., 1997, Hum. Gene Ther.;Wickham, T. J, 2000, Gene Ther. 7, 110–114; Nettelbeck D. M. and Muller;J. V., 2000, Trends Genet. 16, 174–181; Clackson, T., 2000, Gene Ther.7, 120–125; Freundlieb, S, et al., 1999, J. Gene Med. 1, 4–12; Spear M.A., 1998, Anticancer Res. 18, 3223–31, Harvey, D. M. and Caskey C. T.,1998, Curr. Opin. Chem. Biol. 2, 512–518; Clary, B. M. and Lyerly, H.K., 1998, Surg. Oncol. Clin. North Am. 7, 565–574. Luna, M C et al.Cancer Res. 60, 1637–1644; and the references therein.

As mentioned above, more than one carrier and/or targeting molecule orvector may be used simultaneously. For example vectors may be providedin a carrier, e.g. viral vectors such as adenovirus may be carried, eg.in a liposome or polycation structure.

Preferred carriers and vectors for use in the present invention,particularly for use in conjunction with the transfer molecule, includeadenoviruses, polycations such as polylysine (e.g. poly-L-lysine orpoly-D-lysine), polyethyleneimine or dendrimers (e.g. cationicdendrimers such as SuperFect®); cationic lipids such as DOTAP orLipofectin; peptides and targeted vectors such as e.g. transferrinpolylysine or targeted adenovirus vectors. In a particularly preferredembodiment of the invention the carrier is adenovirus.

Such targeting molecules or carriers as described above may also be usedto direct the transfer molecule to particular intracellular compartmentsespecially beneficial for the employment of PCI, for example lysosomesor endosomes.

The intracellular membrane-restricted compartment may be any suchcompartment which is present in a cell. Preferably the compartment willbe a membrane vesicle, especially an endosome or a lysosome. However,the intracellular compartment may also include the Golgi apparatus orthe endoplasmic reticulum.

The light irradiation step to activate the photosensitising agent maytake place according to techniques and procedures well known in the art.For example, the wavelength and intensity of the light may be selectedaccording to the photosensitising agent used. Suitable light sources arewell known in the art. The time for which the cells are exposed to lightin the methods of the present invention may vary. The efficiency of theinternalisation of the transfer molecule into the cytosol appears toincrease with increased exposure to light. A preferred length of timefor the irradiation step depends on the photosensitizer, the amount ofthe photosensitizer accumulated in the target cells or tissue and theoverlap between the absorption spectrum of the photosensitizer and theemission spectrum of the light source. Generally, the length of time forthe irradiation step is in the order of minutes to several hours, e.g.preferably up to 60 minutes e.g. from 0.5 or 1 to 30 minutes, e.g. from0.5 to 3 minutes or from 1 to 5 minutes or from 1 to 10 minutes e.g.from 3 to 7 minutes, and preferably approximately 3 minutes, e.g. 2.5 to3.5 minutes. Appropriate light doses can be selected by a person skilledin the art and again will depend on the photosensitizer and the amountof photosensitizer accumulated in the target cells or tissues. Forexample, the light doses typically used for photodynamic treatment ofcancers with the photosensitizer Photofrin and the protoporphyrinprecursor 5-aminolevulinic acid is in the range 50–150 J/cm² at afluence range of less than 200 mW/cm² in order to avoid hyperthermia.The light doses are usually lower when photosensitizers with higherextinction coefficients in the red area of the visible spectrum areused. However, for treatment of non-cancerous tissues with lessphotosensitizer accumulated the total amount of light needed may besubstantially higher than for treatment of cancers.

Determining the appropriate doses of target molecules for use in themethods of the present invention would be routine practice for a personskilled in the art. Where the transfer molecule is a protein or peptide,for in vitro applications the transfer molecules would generally be usedat doses of less than 5 mg/ml (e.g 0.1–5 mg/ml) and for in vivoapplications the transfer molecules would generally be used at doses ofless than 5 mg/kg (e.g. 0.1–5 mg/kg). Where the transfer molecule is anucleic acid, for in vitro applications an exemplary dose of thetransfer molecules would be approximately 0.1–50 μg nucleic acid per 10⁴cells and for in vivo applications approximately 10⁻⁶–1 g nucleic acidper injection in humans. Where the transfer molecule is associated withan adenovirus carrier, for in vitro applications an exemplary dose wouldbe between 1–1×10⁵ physical virus particles, e.g. 1×10³–1×10⁵ particlesper cell and for in vivo applications the molecule to be introduced inassociation with the adenoviral carrier may be present at aconcentration of 1×10⁻⁹ to 50% such as 3×10⁻⁶ to 50%, e.g. 0.003 to 30%,e.g. 0.2 to 10% (w/w) of virus particles in the final composition foruse in vivo in which w/w refers to the weight of the viral carrier inaddition to the molecule to be introduced relative to the weight of thefinal composition. If used in 1 ml injections, this would correspond toa dose of approximately 10⁵ to 10¹⁵ physical viral particles.

The methods of the invention will inevitably give rise to some cellkilling by virtue of the photochemical treatment i.e. through the actionof the photosensitizing agent. However, this cell death will not matterand may indeed be advantageous for many of the applications (e.g. cancertreatment) and the methods of the invention may be modified such thatthe fraction or proportion of the surviving cells is regulated byselecting the light dose in relation to the concentration of thephotosensitivity agent. Again, such techniques are known in the art.Regardless of the amount of cell death induced by the pure photochemicaltreatment, it is important that the light dose is regulated such thatsome of the individual cells wherein the PCI effect is manifested arenot killed by pure photochemical treatment (although they maysubsequently be killed due to the PCI effect).

In some applications it may be appropriate to retain a larger number ofviable cells after PCI treatment. For example in vaccination and somegene therapy methods viable cells which allow for example antigenpresentation or protein expression is important. In such applications itis appropriate that a population or plurality of cells, substantiallyall of the cells, or a significant majority (e.g. at least 50%, morepreferably at least 60, 70, 80 or 90% of the cells) are not killed. Thisof course is not always desirable especially when PCI is used tointroduce cytotoxic transfer molecules and further cell killing is notdisadvantageous. Cytotoxic effects may also however be achieved by usingfor example gene therapy in which a therapeutic gene is internalizedinto tumour cells by the method of the invention e.g. so that thesecells will produce immunologically active substances that will inducelocal immunological killing of remaining cancer cells or induce asystemic immune response to the tumour cells. In such cases, clearlyafter PCI treatment a proportion of viable cells are required.

The advantages of the present methods and the sequence of treatmentsteps, especially the embodiments wherein the transfer molecule is addedto the cells after the light irradiation step, as compared to thepreviously described methods are

a) photochemical damage to the transfer molecule is diminished;

b) simplification of PCI treatment of internal lesions in combinationwith surgery since photochemical treatment may be performed aftersurgical exposure of the lesion followed by e.g. intratumoral injectionor other local administration of the transfer molecule;

c) the methods are more independent of exact timing of treatment, i.e.the timing of the addition of the molecule to be taken up by the cellsrelative to the time point of illumination. This means that there is agreater “time window” for treatment. This is important since uptake of atherapeutic molecule can vary widely in different clinical situationsand moreover, the uptake is difficult to estimate for individual lesionsin a clinical situation, therefore making a greater time windowextremely advantageous;

d) rapid translocation of the transfer molecule to the cytosol occursthereby substantially decreasing the possibilities for lysosomaldegradation of the transfer molecule.

These advantages are in addition to the advantages associated with PCImethods of internalisation of molecules per se, i.e. 1) there is norestriction on the size of the molecule to be internalised and deliveredto the cytosol as long as the molecule can be endocytosed by the targetcell; 2) the methods are not dependent on cell proliferation; 3) themethods are site specific in that only areas exposed to light areaffected; 4) it is not oncogenic.

The steps of “contacting” the cells with a photosensitising agent andwith the transfer molecule may be carried out in any convenient ordesired way. Thus, if the contacting step is to be carried out in vitrothe cells may conveniently be maintained in an aqueous medium such asfor example appropriate cell culture medium and at the appropriate timepoint the photosensitising agent or transfer molecule can simply beadded to the medium under appropriate conditions, for example at anappropriate concentration and for an appropriate length of time.

The photosensitizing agent is brought into contact with the cells at anappropriate concentration and for an appropriate length of time whichcan easily be determined by a skilled person using routine techniquesand will depend on the particular photosensitizing agent used and thecell type. The concentration of the photosensitizing agent must be suchthat once taken up into the cell, e.g. into, or associated with, one ormore of its intracellular compartments and activated by irradiation, oneor more cell structures are disrupted e.g. one or more intracellularcompartments are lysed or disrupted. For example photosensitising agentsused in the Examples herein may be used at a concentration of forexample 10 to 50 μg/ml. For in vitro use the range can be much broader,e.g. 0.05–500 μg/ml. For in vivo human treatments the photosensitizingagent may be used in the range 0.05–20 mg/kg body weight whenadministered systemically or 0.1–20% in a solvent for topicalapplication. In smaller animals the concentration range may be differentand can be adjusted accordingly.

The time of incubation of the cells with the photosensitizing agent(i.e. the “contact” time) can vary from a few minutes to several hours,e.g. even up to 48 hours or longer. The time of incubation should besuch that the photosensitizing agent is taken up by the appropriatecells.

The incubation of the cells with the photosensitizing agent mayoptionally be followed by a period of incubation with photosensitiserfree medium before the cells are exposed to light or the transfermolecule is added.

The transfer molecule can be any molecule as discussed above and isbrought into contact with the cells at an appropriate concentration andfor an appropriate length of time. Surprisingly it has been found thatthe contact may be initiated even several hours after irradiation. Anappropriate concentration can be determined depending on the efficiencyof uptake of the molecule in question into the cells in question and thefinal concentration it is desired to achieve in the cells. Thus“transfection time” or “cellular uptake time” i.e. the time for whichthe molecules are in contact with the cells can be a few minutes or upto a few hours, for example a transfection time of from 10 minutes untilup to 24 hours, for example 30 minutes up to 10 hours or for example 30minutes until up to 2 hours or 6 hours can be used. An increasedtransfection time usually results in increased uptake of the molecule inquestion. However, the shorter incubation times, for example 30 minutesto 1 hour, seem to result in an improved specificity of the uptake ofthe molecule. Thus, in selecting a transfection time for any method, anappropriate balance must be struck between obtaining a sufficient uptakeof the molecule while maintaining sufficient specificity of uptake.

In vivo an appropriate method and time of incubation by which thetransfer molecules and photosensitizing agents are brought into contactwith the target cells will be dependent on the mode of administrationand the type of transfer molecule and photosensitizing agents. Forexample, if the transfer molecule is injected into a tumour which is tobe treated, the cells near the injection point will come into contactwith and hence tend to take up the transfer molecule more rapidly thanthe cells located at a greater distance from the injection point, whichare likely to come into contact with the transfer molecule at a latertimepoint and lower concentration. In addition, a transfer moleculegiven by intravenous injection may take some time to arrive at thetarget cells and it may thus take longer post-administration e.g.several days, in order for a sufficient or optimal amount of thetransfer molecule to accumulate in a target cell or tissue. The sameconsiderations of course apply to the time of administration requiredfor the uptake of the photosensitizing agent into cells. The time ofadministration required for individual cells in vivo is thus likely tovary depending on these and other parameters.

Nevertheless, although the situation in vivo is more complicated than invitro, the underlying concept of the present invention is still thesame, i.e. the time at which the molecules come into contact with thetarget cells must be such that before irradiation occurs an appropriateamount of the photosensitizing agent has been taken up by the targetcells and either: (i) before or during irradiation the transfer moleculehas either been taken up, or will be taken up after sufficient contactwith the target cells, into different intracellular compartments or (ii)after irradiation the transfer molecule is in contact with the cells fora period of time sufficient to allow its uptake into the cells. Providedthe transfer molecule is taken up into different intracellularcompartments to the photosensitizing agent, the transfer molecule can betaken up before or after irradiation.

The term “cell” is used herein to include all eukaryotic cells(including insect cells and fungal cells). Representative “cells” thusinclude all types of mammalian and non-mammalian animal cells, plantcells, insect cells, fungal cells and protozoa.

The methods of the present invention may be used in vitro or in vivo,either by in situ treatment (for example by utilising targettingmoieties) or by ex vivo treatment followed by the administration of thetreated cells to the body.

The methods of the present invention may be used for example in thetreatment of cancer. Several photosensitisers accumulate preferentiallyin neoplastic tissues, the selectivity for a tumor over the surroundingtissue being usually a factor of 2–3, but this factor may in some cases,such as for brain tissues, be higher, i.e. up to 30. Alternatively, aparticular photosensitising agent may be targeted to a particular tumorby the methods described above. Furthermore, molecules which may be ofclinical interest for treatment of cancer, but are restricted by low orno uptake into the cytosol can be introduced into the cytosol andtargetted to specific cells by means of the present invention. Gelonin,as exemplified below, is an example of such a molecule. Other molecules,either alone or linked to other molecules which target the molecule tobe internalised to a particular cell (e.g. antibodies, transferrin,photosensitisers, apoB on reconstituted LDL-particles) can be used. Theadvantage of such a combination treatment would be 1) enhanced cytotoxiceffect in deeper layers of the tumor tissues since low and subtoxicdoses of light are sufficient for disruption of lysosomes and endosomes;2) enhanced specificity of the treatment since PCI is only given to thetumour area.

Methods of the invention may also be used for treating various otherdisorders, as dictated by the selection of the molecule to be introducedinto the cell, such as rheumatoid arthritis, artherosclerosis and othercardiovascular diseases, virus and other infections, psoriasis, solarkeratosis, wound healing, fracture healing, warts and inherited geneticdisorders such as cystic fibrosis, Gorlin's syndrom and ataxiatelangiectasia.

The methods of the invention may also be used in gene therapy, i.e. thetherapeutic transfer of genes to a patient's cells. Gene therapy ispromising as a method for treating many diseases such as cancer,cardiovascular diseases, viral infections, monogenic disorders such ascystic fibrosis and many other conditions such as those described above.One significant problem in gene therapy today is the high efficiency andspecificity of gene transfer which must occur in vivo. In currentmethods many different carriers or vectors are used for achieving genetransfer in gene therapy. As examples polycationic compounds, cationiclipids and viral systems can be mentioned, but so far in vivo genetherapy has met with little success. Among the many known drawbacks ofthe current methods are low serum stability of the vector, limitedspecificity in gene delivery, low efficiency in gene delivery etc. ThePCI methods of the present invention provide a means of substantiallyimproving both the efficiency and the specificity of many of the genedelivery methods presently employed in gene therapy, by improving thestep of endosomal release which can be efficiency-limiting both for manysynthetic gene delivery vectors and for several viral systems. The lighttreatment inherent in the PCI method also makes it possible to preciselydefine where in the body the enhanced gene transfer shall occur, sincethe increase in gene transfer efficiency will only occur in illuminatedareas. Transfection may be performed, in vitro, in vivo, or ex vivo(with cells or tissues being administered to the patient asappropriate). Preferably suitable carriers and vectors for transfectioninclude adenoviruses, polycations such as polylysine (e.g. poly-D-lysineor poly-L-lysine), SuperFect®, polyethyleneimine or dendrimers; cationiclipids such as DOTAP or Lipofectin or cationic lipids formulated with a“helper lipid” such as DOPE; peptides and targeted vectors such as e.g.transferrin polylysine or targeted adenovirus vectors. In a preferredembodiment of the invention the carrier used for the therapeutic gene isadenovirus.

Another preferred aspect of the present invention is to use non-viralcarrier systems such as for example cationic polymers including peptidesand cationic lipids. Typical polymers include for example polyamine,polyaminoacids including basic polyaminoacids, synthetic and naturalcationic sugar polymers, methacrylate polymers, dendrimers,polyalkylenemines and other polymers known in the art to be useful indrug delivery; especially polymers useful in gene delivery. Typicalcompounds useful according to the present invention include polylysine,polyarginine, poly-L-glutamic acid, polyvinylpyridine, chitosan,polyethylenemine, poly(2-dimethylamino)ethyl methacrylate, histones,protamine, poly(L-ornithine), aviden, spermine, spermidine and anyderivative thereof. In a preferred aspect of the present inventionpolymers described herein may be combined with other polymers orcombined with other gene delivery systems. Non-viral gene deliverysystem useful according to the present invention are, for example,described in R. I. Mahato et al in Advances in Genetics (Eds J. C. Hallet al) (1999) 41 95–156. Cationic polymers are further described in M.C. Garnett in Critical Reviews in Therapeutic Drug Carrier Systems(1999), 16, 147–207, K. A. Howard et al in Biochimica et Biophysica Acta(2000), 1475, 245–255, H. K. Nguyen et al in Gene Therapy (2000), 7,126–138, A. Bragonzi et al in J. Controlled Release (2000), 65, 187–202,S. C. DeSmedt et al in Pharmaceutical Reseach (2000), 17, 113–126 and R.I. Mahoto in J. Drug Targeting (1999), 7, 249–268.

Another aspect of the present invention involves the use of liposomes orother lipid based constructs as non-viral carrier systems. The liposomesmay be pH-sensitive liposomes or non-pH sensitive liposomes.pH-sensitive liposomes consist of at least one pH-sensitive component inthe liposome membrane. Typical compounds include fatty acids such asoleic acid, palmitoylhomocysteine, cholesterol, hemisuccinate morpholinesalt and dieloylsuccinylglyecerol. In addition to the pH-sensitivecomponents, the liposomes may consist ofdioleoylphosphatidylethanolamine (DOPE) and/or other similarphospholipids.

The liposomes or other lipid based delivery system contain preferably atleast one cationic lipid.

The lipid-based delivery system may be present in various types ofaqueous formulation. Various terms are used for these formulations inthe literature: multilamellar liposomes, unilamallar liposomes,pH-sensitive liposomes, nanoemulsions, nanoparticles, proteoliposomes,virosomes, chimerasomes, cochelates, lipofectin® and lipoplex.References to the use of cationic lipids in gene transfer include P. L.Felgner et al in Proc. Natl. Acad. Sci. USA (1987), 84, 7413–7417, D. D.Lasic et al in Adv. Drug Del Rev (1996), 20, 221–266. L. G. Barron et alin Gene Therapy (1999), 6 1179–1183, S. Kawakami et al in PharmaceuticalResearch (2000), 17, 306–313. N. S. Templeton et al in MolecularBiotechnology (1999), 11, 175–180, Y. Zou et al in Cancer Gene Therapy(2000), 7, 683–696, D. D. Stuart et al in Biochemistry et BiophysicaActa (2000), 1463, 219–229, R. I. Mahato et al in Drug Deliv (1997) 4151 and R. J. Lee et al in Crit Rev Drug Carrier Syst (1997), 14, 173.

Lipids useful according to the present invention are, for exampledescribed in U.S. Pat. Nos. 6,120,751, 6,056,938, 6,093,816, 6,039,936,6,034,137, 6,034,135, 6,020,526, 5,980,935, 5,958,935, 5,935,936,5,877,220, 5,830,430, 5,777,153, 5,705,693, 5,459,127, 5,334,761,5,264,618 and references therein.

Typical examples of cationic lipids include N(1-(2,3-dioleyloxy)propyl-N,N,N-trimethyl-ammonium chloride (DOTMA),1,2-dimyristyl-oxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE),1,2-bis(oleoyloxy)-3-(trimethylammino)propane (DOTAP),3β(N′,N′-dimethylaminoethane)-carbamoyl-cholesterol (DC-Chol),2,3-dioleyl-oxy-N[2-sperminecarboxyyl-amido]ethyl-N,N-dimethyl-1-propanaminiumtrifluoracetate (DOS PA), 3-β (N⁴-spermine carbamoyl)-cholesterol,3-β(N⁴-spermidine carbamoyl)-cholesterol and diooctadecylamidoglylspermine (DOGS).

As described above, in one of the preferred embodiments of the inventionthe carrier is cationic lipids. It has earlier been reported thatphotochemical treatment has an inhibiting effect on transfectionmediated by cationic lipids when light is given after the transfermolecule (Prasmickaite et al. (2000), J. Gene Med. 6, in press).However, it has now very surprisingly been shown that when light isgiven before the transfer molecule PCI can have a stimulating effect ontransfection by cationic lipids (see Example 9).

Thus, a further aspect of the invention provides compositions comprisinga transfer molecule and a photosensitizing agent for use in therapy.Optionally the transfer molecule and/or the photosensitizing agent inthe compositions may be associated with carrier molecules such as thosedescribed above. Preferably the compositions are used for cancer therapyor gene therapy. For gene therapy a preferred carrier molecule isadenovirus. Other preferred carriers are cationic lipids.

In a further aspect therefore the present invention provides the use ofa transfer molecule and/or a photosensitizing agent as described hereinfor the preparation of a medicament for use in therapy, wherein saidphotosensitizing agent and separately said transfer molecule iscontacted with cells or tissues of a patient and said cells areirradiated with light of a wavelength effective to activate thephotosensitizing agent and irradiation is performed prior to thecellular uptake of said transfer molecule into an intracellularcompartment containing said photosensitizing agent, preferably prior tocellular uptake of said transfer molecule into any intracellularcompartment. Methods of treatment comprising such methods formalternative aspects of the invention.

Thus, the invention provides a method of treating or preventing adisease, disorder or infection in a patient comprising introducing atransfer molecule into one or more cells in vitro, in vivo or ex vivoaccording to the method as described hereinbefore and where necessary(ie. when transfection is conducted in vitro or ex vivo) administeringsaid cells to said patient.

As defined herein “treatment” refers to reducing, alleviating oreliminating one or more symptoms of the disease, disorder or infectionwhich is being treated, relative to the symptoms prior to treatment.“Prevention” refers to delaying or preventing the onset of the symptomsof the disease, disorder or infection.

As mentioned previously, such methods also have application in methodsof vaccination. Accordingly, a further aspect of the invention providesa method of expressing or presenting an antigenic molecule (the transfermolecule) or a part thereof on the surface of a cell, preferably anantigen-presenting cell, wherein said method comprises the steps asdefined hereinbefore.

As used herein “expressing” or “presenting” refers to the presence ofthe molecule or a part thereof on the surface of said cell such that atleast a portion of that molecule is exposed and accessible to theenvironment surrounding that cell. Expression on the “surface” may beachieved in which the molecule to be expressed is in contact with thecell membrane and/or components which may be present or caused to bepresent in that membrane.

This method may be performed in vitro or in vivo. Preferably however,such antigenic presentation may advantageously result in the stimulationof an immune response, preferably an immune response which confersprotection against subsequent challenge by an entity comprising orcontaining said antigen molecule or part thereof, and consequently theinvention finds particular utility as a method of vaccination.Preferably therefore, the present invention provides a method ofvaccination comprising the method described hereinbefore.

In this aspect of the invention, the transfer molecule as defined hereinis referred to as an “antigenic molecule”. The antigenic molecule may beany molecule wherein that molecule or a part thereof is capable ofstimulating an immune response, when presented to the immune system inan appropriate manner. Advantageously, therefore the antigenic moleculewill be a vaccine antigen or vaccine component, such as a polypeptidecontaining entity.

Many such antigens or antigenic vaccine components are known in the artand include all manner of bacterial or viral antigens or indeed antigensor antigenic components of any pathogenic species including protozoa orhigher orgasms. Whilst traditionally the antigenic components ofvaccines have comprised whole organisms (whether live, dead orattenuated) i.e. whole cell vaccines, in addition sub-unit vaccines, ie.vaccines based on particular antigenic components of organisms e.g.proteins or peptides, or even carbohydrates, have been widelyinvestigated and reported in the literature. Any such “sub-unit”-basedvaccine component may be used as the antigenic molecule of the presentinvention. However, the invention finds particular utility in the fieldof peptide vaccines. Thus, a preferred antigenic molecule according tothe invention is a peptide (which is defined herein to include peptidesof both shorter and longer lengths i.e. peptides, oligopeptides orpolypeptides, and also protein molecules or fragments thereof e.g.peptides of 5–500 e.g. 10 to 250 such as 15 to 75, or 8 to 25 aminoacids). Parts of antigenic molecules which are presented or expressedpreferably comprise parts which are generated by antigen-processingmachinery within the cell. Parts may however be generated by other meanswhich may be achieved through appropriate antigen design (e.g. pHsensitive bands) or through other cell processing means. Convenientlysuch parts are of sufficient size to generate an immune response, e.g.in the case of peptides greater than 5, e.g. greater than 10 or 20 aminoacids in size.

A vast number of peptide vaccine candidates have been proposed in theliterature, for example in the treatment of viral diseases andinfections such as AIDS/HIV infection or influenza, canine parvovirus,bovine leukaemia virus, hepatitis, etc. (see e.g. Phanuphak et al.,Asian Pac. J. Allergy. Immunol. 1997, 15(1), 41–8; Naruse, HokkaidoIgaku Zasshi 1994, 69(4), 811–20; Casal et al., J. Virol., 1995, 69(11),7274–7; Belyakov et al., Proc. Natl. Acad. Sci. USA, 1998, 95(4),1709–14; Naruse et al., Proc. Natl. Sci. USA, 1994 91(20), 9588–92;Kabeya et al., Vaccine 1996, 14(12), 1118–22; Itoh et al., Proc. Natl.Acad. Sci. USA, 1986, 83(23) 9174–8. Similarly bacterial peptides may beused, as indeed may peptide antigens derived from other organisms orspecies.

In addition to antigens derived from pathogenic organisms, peptides havealso been proposed for use as vaccines against cancer or other diseasessuch as multiple sclerosis. For example, mutant oncogene peptides holdgreat promise as cancer vaccines acting an antigens in the simulation ofcytotoxic T-lymphocytes. (Schirrmacher, Journal of Cancer Research andClinical Oncology 1995, 121, 443–451; Curtis Cancer Chemotherapy andBiological Response Modifiers,. 1997, 17, 316–327). A synthetic peptidevaccine has also been evaluated for the treatment of metastatic melanoma(Rosenberg et al., Nat. Med. 1998, 4(3), 321–7). A T-cell receptorpeptide vaccine for the treatment of multiple sclerosis is described inWilson et al., J. Neuroimmunol. 1997, 76(1–2), 15–28. Any such peptidevaccine component may be used as the antigenic molecule of theinvention, as indeed may any of the peptides described or proposed aspeptide vaccines in the literature. The peptide may thus be synthetic orisolated or otherwise derived from an organism.

The cell which is subjected to the methods, uses etc. of this aspect ofthe invention may be any cell which is capable of expressing, orpresenting on its surface a molecule which is administered ortransported into its cytosol.

Since the primary utility of this aspect of the invention resides inantigen-presentation or vaccination, the cell is conveniently an immuneeffector cell i.e. a cell involved in the immune response. However,other cells may also present antigen to the immune system and these alsofall within the scope of this aspect of the invention. The cellsaccording to this aspect are thus advantageously antigen-presentingcells. The antigen-presenting cell may be involved in any aspect or“arm” of the immune response, including both humoral and cell-mediatedimmunity, for example the stimulation of antibody production, or thestimulation of cytotoxic or killer cells, which may recognise anddestroy (or otherwise eliminate) cells expressing “foreign” antigens ontheir surface. The term “stimulating an immune response” thus includesall types of immune responses and mechanisms for stimulating them.

The stimulation of cytotoxic cells or antibody-producing cells, requiresantigens to be presented to the cell to be stimulated in a particularmanner by the antigen-presenting cells, for example MHC Class Ipresentation (e.g. activation of CD8⁺ cytotoxic T-cells requires MHC-1antigen presentation)

Antigen-presenting cells are known in the art and described in theliterature and include for example, lymphocytes (both T and B cells),dendritic cells, macrophages etc. Others include for example cancercells e.g. melanoma cells.

For antigen presentation by an antigen-presenting cell to a cytotoxicT-cell (CTL) the antigenic molecule needs to enter the cytosol of theantigen-presenting cell (Germain, Cell, 1994, 76, 287–299). The presentinvention provides an efficient means of delivery of the antigenicmolecule into the cytosol.

Once released in the cell cytosol by the photochemical internalisationprocess, the antigenic molecule may be processed by theantigen-processing machinery of the cell and presented on the cellsurface in an appropriate manner e.g. by Class I MHC. This processingmay involve degradation of the antigen, e.g. degradation of a protein orpolypeptide antigen into peptides, which peptides are then complexedwith molecules of the MHC for presentation. Thus, the antigenic moleculeexpressed or presented on the surface of the cell according to thepresent invention may be a part or fragment of the antigenic moleculewhich is taken up in the cell.

Antigens may be taken up by antigen-presenting cells by endocytosis anddegraded in the endocytic vesicles to peptides. These peptides may bindto MHC class II molecules in the endosomes and be transported to thecell surface where the peptide-MHC class II complex may be recognised byCD4+ T helper cells and induce an immune response. Alternatively,proteins in the cytosol may be degraded into parts thereof, e.g. byproteasomes and transported into endoplasmic reticulum by means of TAP(transporter associated with antigen presentation) where the peptidesmay bind to MHC class I molecules and be transported to the cell surfaceas illustrated in the FIG. 1 (Yewdell and Bennink, 1992, Adv. Immunol.52: 1–123). If the peptide is of foreign antigen origin, the peptide-MHCclass I complex will be recognised by CD8+ cytotoxic T-cells (CTLs). TheCTLs will bind to the peptide-MHC (HLA) class I complex and thereby beactivated, start to proliferate and form a clone of CTLs. The targetcell and other target cells with the same peptide-MHC class I complex onthe cells surface may be killed by the CTL clone. Immunity against theforeign antigen may be established if a sufficient amount of the antigencan be introduced into the cytosol (Yewdell and Bennink, 1992, supra;Rock, 1996, Immunology Today 17: 131–137). This is the basis fordevelopment of inter alia cancer vaccines. One of the largest practicalproblems is to introduce sufficient amounts of antigens (or parts of theantigen) into the cytosol. This may be solved according to the presentinvention by PCI.

Compositions of the present invention may also comprise a cellcontaining a transfer molecule which has been internalised into thecytosol of said cell by a method of the invention, for use in therapy,particularly cancer therapy, gene therapy and vaccination.

Thus, a yet further aspect of the invention provides a cell or apopulation of cells containing a transfer molecule which has beeninternalised into the cytosol of said cell, which cell is obtainable bya method of the present invention.

A yet further aspect of the invention provides the use of a such a cellor population of cells for the preparation of a composition or amedicament for use in therapy, preferably cancer therapy, gene therapyor vaccination.

The invention further provides a method of treatment of a patientcomprising administering to said patient cells or compositions of thepresent invention, ie. a method comprising the steps of introducing amolecule into a cell as described hereinbefore and administering saidcell thus prepared to said patient. Preferably said methods are used totreat cancer, in gene therapy or for vaccination.

In vivo, any mode of administration common or standard in the art may beused, e.g. injection, infusion, topical administration, both to internaland external body surfaces etc. For in vivo use, the invention can beused in relation to any tissue which contains cells to which thephotosensitising agent and the transfer molecule are localized,including body fluid locations, as well as solid tissues. All tissuescan be treated as long as the photosensitiser is taken up by the targetcells, and the light can be properly delivered.

Thus, the compositions of the invention may be formulated in anyconvenient manner according to techniques and procedures known in thepharmaceutical art, e.g. using one or more pharmaceutically acceptablecarrier or excipients. “Pharmaceutically acceptable” as referred toherein refers to ingredients that are compatible with other ingredientsof the compositions as well as physiologically acceptable to therecipient. The nature of the composition and carriers or excipientmaterials, dosages etc. may be selected in routine manner according tochoice and the desired route of administration, purpose of treatmentetc. Dosages may likewise be determined in routine manner and may dependupon the nature of the molecule, purpose of treatment, age of patient,mode of administration etc. In connection with the photosensitizingagent the potency/ability to disrupt membranes on irradiation, shouldalso be taken into account.

The invention will now be described in more detail in the followingnon-limiting Examples with reference to the following drawings in which:

FIG. 1 shows light-induced transfection of THX cells with a pEGFP-N1polylysine complex, wherein the cells are contacted with the pEGFP-N1polylysine complex before or after the cells are exposed to light asindicated in the Figure.

FIG. 2 shows light-induced transfection of HCT-116 cells with a pEGFP-N1polylysine complex, wherein the cells are contacted with the pEGFP-N1polylysine complex before or after the cells are exposed to light asindicated in the Figure.

FIG. 3 shows light-induced treatment of THX cells with gelonin and thusa reduction in protein synthesis, wherein the cells are contacted withthe gelonin molecule before or after the cells are exposed to light asindicated in the Figure.

FIG. 4 shows the effect on the efficiency of light-induced transfectionof THX cells of contacting the cells with the pEGFP-N1 polylysinecomplex immediately after light exposure and at later timepoints afterlight exposure.

FIG. 5 shows the effect on the efficiency of light-induced transfectionof THX cells when the cells are contacted with the pEGFP-N1 polylysinecomplex at varying timepoints before and after light exposure.

FIG. 6 shows the effect on light-induced transfection of THX cells whenthe cells are contacted with the pEGFP-N1 polylysine complex for variouslengths of time after light exposure.

FIG. 7 shows a schematic representation of a potential model by whichthe current invention may work.

A. I. The photosensitizer S is endocytosed (I) and ends up inintracellular vesicles (II). These vesicles rupture upon exposure tolight (III).

B. After the photochemical treatment as described in A the cells aretreated with a molecule M which is endocytosed and ends up inintracellular vesicles. These vesicles will fuse with photochemicallydamaged vesicles and the molecule M will be released into the cytosol.

FIG. 8 shows the effect on transfection of treatment of the cells at 0°C. with the pEGFP-N1 polylysine complex.

FIG. 9 shows light-induced transfection of pEGFP-N1 polylysine complexwhen 3-THPP, which is mainly not localized in endocytoxic vesicles, isused as a photosensitizing agent.

FIG. 10 shows the effect of a combination of photosensitizer and lightpretreatment on the transfection of cells with cationic lipids.

FIG. 11 shows the effect of photochemical treatment on adenovirustransduction of THX cells.

FIG. 12 shows the effect of photochemical treatment on intracellularlocalisation of FITC-dextran. THX cells were incubated with 20 μg/mlAlPcS_(2a) for 18 h followed by 4 h incubation in AlPcS_(2a)-freemedium. Then the cells were either exposed to light for 4 min (B,D) orkept in the darkness (A,C) before a 3 h incubation with 5 mg/mlFITC-dextran. Fluorescence (A,B) and phase contrast (C,D) micrographs.

FIG. 13 shows the effect of photochemical treatment on gelonin toxicityin THX and HCT 116 cells. For the “light before” strategy, the cellswere first incubated with 20 μg/ml AlPcS_(2a) for 18 h, then for another4 h in AlPcS_(2a)-free medium before exposure to light as indicated inthe figure. After illumination 1 μg/ml gelonin was added, and the cellswere incubated for 18 h. For the “light after” strategy the cells wereco-incubated with 20 μg/ml AlPcS_(2a) and 1 μg/ml gelonin for 18 hbefore exposure to light as indicated in the figure. The control cellswere treated only with 1 μg/ml gelonin for 18 h and exposed to light; oronly with 20 μg/ml AlPcS_(2a) for 18 h, chased 4 h in AlPcS_(2a)-freemedium and exposed to light. [³H]-leucine incorporation into proteinswas measured the next day after light treatment and expressed asrelative protein synthesis. Data points represent mean±standard error(S.E.) of triplicates.

FIG. 14 shows the effect of photochemical treatment on expression ofβ-galactosidase in THX cells infected with AdHCMV-lacZ. For the “lightbefore” strategy AlPcS_(2a)-pretreated cells were incubated for another4 h in AlPcS_(2a)-free medium before light exposure for 3 min. Followingillumination the cells were infected with AdHCMV-lacZ (at MOI 1) for 30min at 37° C. Then 2 ml of medium was added and the cells were incubatedfor two days before analysis of β-galactosidase expression. For the“light after” strategy AlPcS_(2a)-treated cells were incubated inAlPcS_(2a)-free medium for 3 h before a 30 min infection withAdHCMV-lacZ. After addition of 2 ml of culture medium the cells wereincubated for another 30 min before illumination for 3 min, and two dayslater were analysed for β-galactosidase expression.

FIG. 15 shows the effect of the incubation time on the efficiency oflight-induced transfection with pEGFP/polylysine in THX and HCT 116cells. AlPcS_(2a)-pretreated cells were washed and incubated inAlPcS_(2a)-free medium for 4 h before light exposure for 3 min (THXcells) or 7 min (HCT) cells. Following illumination thepEGFP-N1/polylysine complex (5 μg/ml plasmid) was added, and the cellswere incubated for different time periods indicated in the figure. Afterremoving the complex fresh complex-free medium was added and the cellswere incubated for two days before analysis of EGFP expression.

FIG. 16 shows the effect of the light first PCI strategy on transfectionof THX cells mediated by poly-L-lysine using TPPS_(2a) as thephotosensitiser compared to the light after strategy, using variousirradiation times. PLL-L: Irradiation after the pEGFP-N1/PLL complex.L-PLL: Irradiation before the pEGFP-N1/PLL complex. Unshaded bars—nolight, shaded bars—70 seconds irradiation, solid bars—100 secondsirradiation.

FIG. 17 shows the effect of the light first PCI strategy on transfectionof THX cells mediated by poly-L-lysine using TPPS₄ as thephotosensitiser compared to the light after strategy, using variousirradiation times. PLL-L: Irradiation after the pEGFP-N1/PLL complex.L-PLL: Irradiation before the pEGFP-N1/PLL complex. Unshaded bars—nolight, horizontally shaded bars—50 seconds irradiation, verticallyshaded bars—70 seconds irradiation, solid bars—100 seconds irradiation.

FIG. 18 shows the effect of the light first PCI strategy on transfectionof HCT 116 cells mediated by poly-L-lysine, using TPPS_(2a) as thephotosensitiser compared to the light after strategy, using variousirradiation times. PLL-L: Irradiation after the pEGFP-N1/PLL complex.L-PLL: Irradiation before the pEGFP-N1/PLL complex. Unshaded bars—nolight, shaded bars—70 seconds irradiation, solid bars—100 secondsirradiation.

FIG. 19 shows the effect of the light first PCI strategy on transfectionof HCT 116 cells mediated by poly-L-lysine, using TPPS₄ as thephotosensitiser. PLL-L: Irradiation after the pEGFP-N1/PLL complex.L-PLL: Irradiation before the pEGFP-N1/PLL complex. Unshaded bars—nolight, horizontally shaded bars—1.5 minutes irradiation, verticallyshaded bars—2 minutes irradiation, solid bars—3 minutes irradiation.

FIG. 20 shows the effect of the light first PCI strategy on transfectionmediated by DOTAP using TPPS₄ as the photosensitiser compared to thelight after strategy, using various irradiation times. DOTAP-L:Irradiation after the pEGFP-N1/DOTAP complex. L-DOTAP: Irradiationbefore the pEGFP-N1/DOTAP complex. Unshaded bars—no light, shadedbars—70 seconds irradiation, solid bars—100 seconds irradiation.

FIG. 21 shows the effect of the light first PCI strategy on transfectionmediated by SuperFect® using TPPS_(2a) as the photosensitiser, wherevarious illumination and transfection times are used and the amount ofDNA for transfection varied. ▪—transfection for 1 hour with 0.75 μg DNA;▴—transfection for 4 hours with 0.75 μg DNA; ∘—transfection for 1 hourwith 1.5 μg DNA.

FIG. 22 shows the effect of the light first PCI strategy on adenovirusmediated gene transduction of HCT 116 cells using AlPcS_(2a) as thephotosensitiser, in which the virus is added at various times. Thepercentage of transduced cells when virus was added at different timepoints was analysed by flow cytometry as described in the Example. Thetime points of addition of the virus complexes are indicated on thefigure. Time points to the left of the Y-axis represent virus addedbefore irradiation, time points to the right represent addition of virusafter irradiation. ▪—1 minute irradiation; □—no irradiation.

FIG. 23 shows the effect of the light first PCI strategy on transfectionof HCT 116 cells mediated by Poly-D-lysine using A1PcS_(2a) as thephotosensitiser, with variable irradiation times.

FIG. 24 shows the effect of the light first PCI strategy on cell killingby the cytostatic agent bleomycin using TPPS_(2a) as thephotosensitiser, with variable amounts of bleomycin, irradiation timesand transfection times. ⋄—5 TPPS−, 1 h=5 μM bleomycin, withoutTPPS_(2a), 1 h incubation; ♦—5 TPPS+, 1 h=5 μM bleomycin, withTPPS_(2a), 1 h incubation; Δ—25 TPPS−, 1 h=25 μM bleomycin, withoutTPPS_(2a), 1 h incubation; ▴—25 TPPS+, 1 h=25 μM bleomycin, withTPPS_(2a), 1 h incubation; □—100 TPPS−, 1 h=100 μM bleomycin, withoutTPPS_(2a), 1 h incubation; ▪—100 TPPS+, 1 h=100 μM bleomycin, withTPPS_(2a), 1 h incubation; ∘—100 TPPS−, 4 h=100 μM bleomycin, withoutTPPS_(2a), 4 h incubation; ●—100 TPPS+, 4 h=100 μM bleomycin, withTPPS_(2a), 4 h incubation.

FIG. 25 shows the effect of PCI with gelonin for treatment of tumours ina mouse in vivo model. The treatment groups were as follows: (Δ) geloninonly; (▪) a placebo treatment of phosphate buffered saline (PBS)injection combined with illumination; (⋄) only the photochemicaltreatment (i.e. AlPcS_(2a)+light), but no gelonin; (●) full gelonin PCItreatment (i.e. AlPcS_(2a)+gelonin+light).

EXAMPLES Materials and Methods

Cell Lines

The human melanoma cell line THX was established from tumour tissueobtained from a patient treated for metastatic malignant melanoma at theNorwegian Radium Hospital (Aamdal et al., 1986., Int. J. Cancer, 37,579), and grown in RMPI 1640 (Gibco-BRL) supplemented with 10% FCS(Gibco-BRL) and 2 mM glutamine (Gibco-BRL). The human colon carcinomacell line HCT 116 was obtained from American Type Culture Collection(ATCC no. CCL-247) and grown in RPMI 1640 medium supplemented with 10%foetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mMglutamine (all Gibco BRL, Paisley, UK).

Irradiation

Two different light sources were used for treatment of the cells, bothconsisting of a bank of 4 fluorescent tubes. Cells treated with TPPS₄,TPPS_(2a), and 3-THPP (Porphyrin Products, Logan, Utah) were exposed toblue light (model 3026; Appl. Photophysics, London, UK) with a lightintensity reaching the cells of 1.5 mW/cm² while cells treated withAlPcS_(2a) (Porphyrin Products, Logan, Utah) were exposed to red light(Philips TL 20W/09) filtered through a Cinemoid 35 filter with a lightintensity reaching the cells of 1.35 mW/cm².

Fluorescence Microscopy

The cells were analysed by fluorescence microscopy as described in Berg.K., et al., Biochem. Biophys. Acta., 1370: 317–324, 1998. For analysisof fluorescein-labelled molecules the microscope was equipped with a450–490 nm excitation filter, a 510 nm dichroic beam splitter and a510–540 nm band pass emission filter.

Preparation of Plasmid-pLys Complexes and Treatment of Cells

Plasmid-pLys complexes (charge ratio, 1.7 as described in Berg et al.(1999) Cancer Res. 59: 1180–83) were prepared by gently mixing 5 μgplasmid (pEGFP-N1; Clontech Laboratories, Inc., Palo Alto, Calif.) in 75μl of HBS with 5.3 μg pLys (MW 20700; Sigma, St. Louis, Mo.) in 75 μl ofHBS. The solutions were incubated for 30 min at room temperature,diluted with culture medium and added to the cells.

THX cells were incubated with 20 μg/ml AlPcS_(2a) for 18 hours at 37°C., washed and incubated in sensitizer-free medium for 3 hours beforeincubation with plasmid-pLys complexes for 1 hour followed by exposureto light. Alternatively, after the AlPcS_(2a) incubation the cells werewashed and incubated in sensitizer free medium for 4 hours beforeexposure to light followed by a 1 hour incubation with the plasmid-pLyscomplexes. The cells were incubated at 37° C. for 2 days, beforeanalysis of GFP expression was carried out by flow cytometry.

HCT-116 cells were incubated with 20 μg/ml AlPcS_(2a) for 18 hours,washed incubated for 4 hours in the absence of AlPcS_(2a) before lightexposure. The cells were treated with pEGFP-N1 polylysine complex for 4hours immediately before or after exposure to light. After 2 daysincubation at 37° C. the GFP expression was studied by flow cytometry.

Flow Cytometry Analysis

The cells were trypsinized, centrifuged, resuspended in 400 μl ofculture medium and filtered through a 50 μm mesh nylon filter. Then thecells were analyzed by a FACS-Calibur (Becton Dickinson) flow cytometer.For each sample 10000 events were collected. Fluorescein-fluorescence(for example green Fluorescent protein (GFP)) was measured through a510–530 nm filter after excitation with an argon laser (200 mW) tuned on488 nm. AlPcS_(2a) was measured through a 670 nm longpass filter afterexcitation with a diode laser (50 mW) tuned on 635 nm. Cell doublets anddead cells were discriminated from single viable cells by gating. Thedata were analysed with CELLQuest Software (Becton Dickinson).

Example 1

Light-Induced Transfection as a Function of Light Dose

THX cells were treated with 20 μg/ml AlPcS_(2a) for 18 hours, washed andincubated in sensitizer-free medium for 3 hours followed by 1 hourincubation with 5 μg pEGFP-N1/polylysine complex before light exposurefor 1, 2, 3 or 4 minutes. Alternatively, after the AlPcS_(2a) incubationthe cells were washed and incubated for 4 hours in sensitizer-freemedium before light treatment for 1, 2, 3 or 4 minutes followed by 1hour with pEGFP-N1/polylysine complex as indicated on FIG. 1. GFPexpression was analysed by flow cytometry 48 hours after light exposure.The charge ratio for pEGFP-N1/polylysine complex was 1.7.

The results are shown in FIG. 1 and it can be seen that the transfectionof GFP is equally as effective when the plasmid-pLys complex (i.e.pEGFP-N1/pLys) is added to the cells after rather than before lightexposure. It can also be seen that with both treatments the percentageof transfected cells depends on the length of time the cells are exposedto light with the percentage reaching a maximum level at around 2minutes and then levelling off.

Example 2

Expression of GFP in HCT-116 Cells

HCT-116 cells were incubated with 20 μg/ml AlPcS_(2a) for 18 hoursfollowed by 4 hours in the absence of AlPcS_(2a) before exposure tolight. The cells were treated with pEGFP-N1/polylysine complex for 4hours immediately before or after exposure to light as indicated on FIG.2. The expression of GFP was measured by flow cytometry 2 days after thelight exposure.

The results are shown in FIG. 2 and it can be seen that, in a similarway to the transfection of the THX cells in Example 1, the transfectionof GFP is equally as effective when the plasmid-pLys complex (i.e.pEGFP-N1/pLys) is added to the cells after rather than before lightexposure. Again, the percentage of transfected cells varies depending onthe length of time the cells are exposed to light.

Example 3

Synergistic Effects of Adding Gelonin Before and After PhotochemicalTreatment

Gelonin is a plant toxin which efficiently inhibits protein synthesiswhen it is present in the cytosol of cells. THX cells were incubatedwith 20 μg/ml AlPcS_(2a) for 18 hours followed by 4 hours insensitizer-free medium before exposure to light. The cells were eitherco-treated with AlPcS_(2a) and 1 μg/ml gelonin, or gelonin (1 μg/ml) wasadded to the medium immediately after light exposure for 18 hours afterwhich it was removed from the medium as indicated on FIG. 3. Proteinsynthesis was measured 24 hours after light exposure.

The results are shown in FIG. 3 and it can be seen that although thephotochemical treatment itself in the absence of gelonin leads to somereduction in protein synthesis, the presence of gelonin either before orafter photochemical treatment induces a significantly greater inhibitionof protein synthesis. This data shows that gelonin is internalised intothe cells whether the gelonin is contacted with the cells before orafter photochemical treatment.

Example 4

Effect of Chase Time on Light-Induced Transfection

THX cells were treated with 20 μg/ml AlPcS_(2a) for 18 hours, washed andincubated in sensitizer-free medium for 4 hours before 3 minutes oflight treatment. The cells were incubated in growth medium for the timesindicated on FIG. 4 before treatment with pEGFP-N1/polylysine complex(charge ratio 1.7) for 1 hour. GFP expression was analysed by flowcytometry 48 hours after light exposure.

The results are shown in FIG. 4 and it can be seen that for the bestresults the molecule to be internalised should be exposed to the cellsrelatively soon after the photochemical treatment, since thetransfection with pEGFP-N1 declines with a half life of about 5 hoursafter light exposure.

Example 5

Efficiency of Transfection as a Function of a Transfection PulseRelative to Illumination

THX cells were treated with 20 μg/ml AlPcS_(2a) for 18 hours, washed andincubated in sensitizer-free medium. Cells were given a pulse (0.5 or 1hour, the width of the bar on FIG. 5 reflects the beginning and end oftreatment) of treatment with the pEGFP-N1/polylysine complex eitherbefore (negative bascissa values) or after (positive abscissa values) 3minutes of light exposure. GFP expression was analysed by flow cytometry48 hours after light exposure. Data from several experiments have beennormalized taking the efficiency of transfection when transfection isperformed just before or just after light exposure as 100%.

The results are shown in FIG. 5 and it can be seen that for the besttransfection efficiency the cells should be exposed to the molecules tobe internalised either shortly before or after exposure to light.

Example 6

Light-Induced Transfection—Dependence on Incubation Time withpEFGP-N1/Polylysine Complex

THX cells were treated with 20 μg/ml AlPcS_(2a) for 18 hours, washed andincubated in sensitizer-free medium for 4 hours before exposure tolight, followed by incubation with pEGFP-N1/polylysine complex (chargeratio 1.7) for up to 6 hours as illustrated in FIG. 6A. GFP expressionwas analysed by flow cytometry 48 hours after light exposure. The numberof GFP-expressing cells after such treatments are presented in FIG. 6Band the specificity of the different treatments presented in FIG. 6C.

The results are shown in FIG. 6 and it can be seen that although thenumber of transfected cells increases with increasing incubation timewith the pEGFP-N1/polylysine complex (FIG. 6B), the highest specificityof transfection occurs after the shortest incubation times (FIG. 6C).

Example 7

Treatment with pEGFP-N1/Polylysine Complex at 0° C.

This experiment was designed to test whether the photochemical treatmentled to minor damage to the cell membranes, thereby meaning that theplasmid brought into contact with the cells after light treatment couldleak through the plasma membrane.

THX cells were treated with 20 μg/ml AlPcS_(2a) for 18 hours, washed andincubated in sensitizer-free medium for 4 hours before light exposurefollowed immediately afterwards by 45 minutes incubation at 0° C. withpEGFP-N1/polylysine complex (charge ratio 1.7). The cells were theneither A) trypsinised and seeded out before being transferred to 37° C.,or B) transferred to 37° C. without trypsinization. The cells wereexposed to light as indicated on FIG. 8. GFP expression was analysed byflow cytometry 48 hours after light exposure.

After the incubation of the cells for 45 minutes at 0° C. withpEGFP-N1/polylysine complex the complex will stick to the cell surfacebut not be endocytosed. The THX cells were then either incubated inplasmid-free medium at 37° C. (FIG. 8B) or trypsinised (FIG. 8A) toremove the plasmid from the surface and seeded out in new dishes at 37°C. These experiments indicate that the plasmid/polylysine complex doesnot leak through the plasma membrane after photochemical treatment.

Example 8

Combination of 3-THPP and Light with Treatment with pEGFP-N1/PolylysineComplex

THX cells were treated with 0.25 μg/ml 3THPP for 18 hours, washed andincubated in sensitizer-free medium for 4 hours before light exposure,followed immediately afterwards by 1 hour incubation withpEGFP-N1/polylysine complex (charge ratio 1.7). The cells were exposedto light as indicated on FIG. 9 and analysed for GFP-expression flowcytometrically 48 hours after light exposure.

3-THPP is a photosensitizer the main location of which is not inendosomes or lysosomes. The results shown in FIG. 9 indicate thattreatment of cells with 3-THPP before irradiation induces only a minorincrease in GFP expression in comparison to the results shown inprevious examples where the photosensitizing agent AlPcS_(2a) is used.This indicates that a photosensitizing agent that is localised inendosomes and lysosomes may be advantageous.

Example 9

Combination of Photosensitizer and Light Pretreatment AllowsTransfection of Cells Using Cationic Lipids

HCT 116 cells were seeded out at a density of 75 000 cells/well in a12-well plate one day before the experiment. The cells were incubatedwith the photosensitizer AlPcS_(2a) (20 ug/ml) for 18 hours followed bya 7 h-chase in photosensitizer-free medium, and exposed to red light for7 min. Then the cells were incubated with a DOTAP-complex (DOTAP waspurchased from Boehringer) with the plasmid pEGFP-N1 (5:1 DOTAP/plasmid,1 ug/ml pEGFP-N1) for 3 h, washed with the growth medium and incubatedat 37° C. for 21 h before the expression of EGFP was measured by flowcytometry as described under Materials and Methods. Control cells werenot exposed to light, otherwise the treatment was identical.

The results are shown in FIG. 10 and it can be seen that thePCI-treatment increases the transfection efficiency with theDOTAP/plasmid complex about 4 times.

Example 10

Effect of PCI on Adenovirus Transduction of THX Cells

Material

Fluorescein di-β-D-galactopyranoside (FDG) was purchased from MolecularProbes (F-1179). A 20 mM stock solution was prepared by dissolving thepowder in a 1:1 mixture of DMSO/ethanol. The mixture was gradually addedto an appropriate volume ice-cold water to make a 8:1:1 H₂0/DMSO/ethanolsolution.

The recombinant virus AdCA171acZ was formed and propagated in the humancell line 293, an Ad E1-transformed embryonic kidney cell linemaintained in MEM F-11 medium supplemented with 10% FCS, 100 U/mlpenicillin (Gibco-BRL), 0.1 mg/ml streptomycin (Gibco-BRL) and 2 mMglutamine.

Construction of Recombinant Virus

The recombinant adenovirus AdCA171acZ encoding the E. coli lacZ geneunder control of the human CMV promoter was obtained by homologousrecombination using the pJM17 system in 293 cells (Addison et al., 1997,J. Gen. Virol., 78, 1653–1661). Recombinant vectors were plaquepurified, grown to high titre in 293 cells and purified by cesiumchloride banding as previously described (Hitt et al., 1995, Methods inMol. Genetics., 7, 15–30).

Sensitizing of Cells

The THX cells (4×10⁵ cells) were seeded out in 6 cm dishes and allowedto grow overnight. At approximately 60% confluence the growth medium wasexchanged with 2 ml growth medium supplemented with 20 μg/ml AlPcS_(2a),and the dishes were placed back into the incubator for 16–18 hours. Thesensitizer-containing medium was then sucked off, and the cells wereincubated in ordinary growth medium at least 4 hours before lighttreatment and virus infection.

Infection of Cells

Trypsin-EDTA was used to detach cells from three dishes and the meancell number in the dishes was calculated by Bürcher chamber counting.Adenovirus dilutions were prepared in PBS with 0.68 MM CaCl₂ and 0.5 mMMgCl₂ according to the number of cells to infect. Usually the cells wereinfected at an m.o.i. (multiplicity of infection) of 1 and 10.

Before virus was added the cells were exposed to red light (Philips TL20W/09, filtered through a Cinemoid 35 filter with a light intensityreaching the cells of 1.35 mW/cm²) for 3 minutes. Subsequently themedium was sucked off and 200 μl virus suspension (or PBS with 0.68 mMCaCl₂ and 0.5 mM MgCl₂ in the cases of controls not treated with virus)was added to each dish. After incubation for 30 minutes at 37° C., 5 mlordinary growth medium was added and the cells were allowed to grow for48 hours.

β-galactosidase Assay

The cells were detached by Trypsin-EDTA and resuspended in 5 ml growthmedia. After centrifugation for 5 minutes at 1000 rpm, the medium wassucked off, the cell pellets resuspended in 50 μl growth medium and thetubes placed in a 37° C. water bath for 5 minutes. Subsequently, 50 μlof 2 mM FDG-solution preheated to 37° C. was added and the tubes placedback into the water bath for 1 minute. Finally, 900 μl growth medium wasadded and the tubes were incubated on ice for 30–60 minutes before thesamples were analysed by flow cytometry as described above.

THX cells were treated with AlPcS_(2a) (denoted as PS on FIG. 11) andadenovirus (denoted as “virus” on FIG. 11) and exposed to 3 or 4 minutesof light as described in Material and Methods and measured forβ-galactosidase (β-gal) activity by flow cytometry. The total β-galactivity was quantified by integrating the β-gal positive cells andtheir β-gal activity. Both the number of β-gal positive cells and themean β-gal activity was increased by the PCI treatment.

The results show that minimal infection of THX cells occurs when thecells are incubated with virus alone or virus and photosensitising agentbut that photochemical treatment, i.e. the addition of light to thephotosensitising agent significantly potentiates the transduction ofcells (as shown by the increase in β-gal activity).

Example 11

Effect of Photochemical Treatment on the Intracellular Localisation ofan Endocytosis Marker Molecule.

THX cells were seeded out into Falcon 3001 dishes (2.5×10⁴ cells perdish) and the next day treated with 20 μg/ml AlPcS_(2a) for 18 h, washedfrom AlPcS_(2a) and incubated in AlPcS_(2a)-free medium for 4 h. Thenthe cells were exposed to light for 4 min before a 3 h incubation with 5mg/ml of the endocytic marker FITC-dextran. Non-illuminated cells weretreated in a similar way except for illumination. The intracellularlocalisation of FITC-dextran in unfixed cells was observed with a ZeissAxioplan fluorescence microscope (Oberkochen, Germany) using anobjective with 63× magnification, a 450–490 nm band pass excitationfilter and a 510–540 band pass emission filter. Fluorescence micrographswere recorded by means of a cooled charge-coupled device (CCD) camera(Photometrics Inc., Tucson, Ariz.).

The results show (FIG. 12) that PCI with light given before thefluorescent endocytic marker FITC-dextran shifts the localisation ofthis marker from endocytic vesicles (the spots seen in panel A fornon-illuminated cells) to the cytosol (the diffuse fluorescence seen inpanel B for illuminated cells). Thus when the light treatment is givenbefore the macromolecule to be internalised the macromolecule is veryrapidly translocated to the cytosol, substantially decreasing thepossibilities for lysosomal degradation of the macromolecule.

Example 12

Effects of Photochemical Treatments on Gelonin Toxicity in THX- and HCT116 Cells

Gelonin is a plant toxin that efficiently inhibits protein synthesiswhen it is present in the cell cytosol, but which is not able to reachthe cytosol on its own, and therefore is quite non-toxic to intactcells. For the treatment with gelonin 25×10³ cells per well were seededout into 24-well plates (Nunc, Denmark). The next day 20 μg/mlAlPcS_(2a) was added, and the cells were incubated for 18 h at 37° C.All the procedures after AlPcS_(2a) addition were carried out in subduedlight. For the “light before” strategy, the cells were washed fromAlPcS_(2a) and incubated in AlPcS_(2a)-free medium for 4 h. Then thecells were exposed to light (as indicated in the figures) before thetreatment with 1 μg/ml gelonin for 18 h. For the “light after” strategythe cells were co-incubated with 20 μg/ml AlPcS_(2a) and 1 μg/ml geloninfor 18 h, and washed before exposure to light as indicated in thefigure.

Non-illuminated cells were treated in a similar way except forillumination. The treated cells were washed once with culture medium andafter addition of fresh medium incubated at 37° C. before furtheranalysis. Inhibition of protein synthesis was assayed by [³H]-leucineincorporation into protein 24 h after light exposure. Illumination wasperformed from a bench with four light tubes (Philips TL 20W/09) and along pass filter with a cut off at 550–600 nm. The light intensityreaching the cells was 13.5 W/m².

The example shows that in both THX- (FIG. 13A) and HCT 116 (FIG. 13B)cells the “light before” strategy works better than the “light after”method. Thus, for THX-cells at the highest light dose the inhibition ofprotein synthesis was about 3-fold more potent with “light before” thanwith “light after”. It can also be seen that in both cell lines geloninin itself had no toxic effect without the PCI treatment, showing thepotency and specificity in the induction of the toxin effects that canbe achieved by the photochemical treatment.

Example 13

Photochemical Stimulation of Adenovirus-Mediated Gene Transduction

5×10⁴ THX cells per well were seeded out into 6-well plates. The nextday 20 μg/ml AlPcS_(2a) was added, and the cells were incubated for 18 hat 37° C. All the procedures after AlPcS_(2a) addition were carried onin subdued light. For the “light before” strategy, the cells were washedfrom AlPcS_(2a) and incubated in AlPcS_(2a)-free medium for 4 h. Thenthe cells were exposed to light for 3 min before the treatment with theadenoviral vector AdHCMV-lacZ (also referred to in Example 10 asAdCA171acZ) at a multiplicity of infection (MOI) of 1 for 30 min. Thisvector contains a β-galactosidase reporter gene whose expression can beanalysed by flow cytometry (see below).

For the “light after” strategy AlPcS_(2a)-treated and washed cells werefirst treated with adenovirus at the same concentration and for the sametime as indicated above, washed, and after addition of fresh culturemedium exposed to light. Non-illuminated cells were treated in a similarway except for illumination.

The treated cells were washed once with culture medium and afteraddition of fresh medium incubated at 37° C. before further analysis.β-galactosidase expression was analysed by flow cytometry two days afterlight exposure. Detailed methods for construction of the virus (which isreferred to either as AdHCMV-lacZ or AdCA171acZ), treatment of thecells, illumination and analysis of β-galactosidase expression aredescribed under Example 10.

The results (FIG. 14) show that the photochemical treatment using the“light before” procedure (shown by the bars on the right hand side ofFIG. 14) increases the percentage of β-galactosidase-expressing cellsabout 6-fold; from 2.5% to 15% under these experimental conditions. Itcan also be seen that the effect with the “light before” procedure wasalmost equal to what was obtained with the “light after” method (shownby the bars on the left hand side of FIG. 14).

Example 14

Effect of the Incubation Time on the Efficiency of Light-InducedTransfection

5×10⁴ THX cells or 7.5×10⁴ HCT 116 cells per well were seeded out into6-well and 12-well plates, respectively. The next day 20 μg/mlAlPcS_(2a) was added, and the cells were incubated for 18 h at 37° C.All the procedures after AlPcS_(2a) addition were carried on in subduedlight. The cells were washed from AlPcS_(2a) and incubated inAlPcS_(2a)-free medium for 4 h. Then the cells were exposed to light (3min for THX cells or 7 min for HCT 116 cells) before treatment withpEGFP-N1/polylysine (5 μg/ml pEGFP-N1) complex for the times indicatedon FIG. 15. Non-illuminated cells were treated in a similar way exceptfor illumination. The treated cells were washed once with culture mediumand after addition of fresh medium incubated at 37° C. for 2 days beforeanalysis of EGFP expression by flow cytometry (see Materials andMethods). Illumination was performed from a bench with four light tubes(Philips TL 20W/09) and a long pass filter with a cut off at 550–600 nm.The light intensity reaching the cells was 13.5 W/m².pEGFP-N1/polylysine complex (charge ratio 1.7) was prepared by gentlymixing plasmid and polylysine solutions prepared separately: 5 μgpEGFP-N1 plasmid was diluted in 75 μl water and 5.3 μg polylysinediluted in 75 μl water. The solutions were mixed and incubated for 30min at room temperature, diluted with culture medium to 1 ml and addedto the cells.

The results (FIG. 15) show that both in THX and in HCT 116 cellstransfection by DNA/polylysine complexes can be strongly induced by the“light before” photochemical treatment. It can be seen that thestimulation of transfection is effective already after short incubationstimes with DNA, at least down to 30 min incubation time. Thelight-induced transfection increases with the incubation time, however,seemingly levelling off after about 2 h incubation with DNA.

Example 15

Effect of the Light First PCI Strategy on Transfection of THX CellsMediated by poly-L-lysine Using TPPS_(2a) as the Photosensitiser

Tetraphenylporphine disulfonate (TPPS_(2a)), lot #04197, was produced byPorphyrin Products (UT, USA). TPPS_(2a) was dissolved in DMSO.

The plasmid pEGFP-N1 was purchased from Clontech Laboratories Inc. (CA,USA; cat. no. 6085–1). The batch used (lot# EGFP-N1-1002) was producedby ELIM Biopharmaceuticals, Inc. (CA, USA) and delivered at aconcentration of 5 mg/ml in sterile water. A stock solution of 0.5 mg/mlwas made up in sterile TE-buffer pH 7.4 (1 mM Tris-HCl, 1 mM EDTA) andkept at −20° C.

The THX human melanoma cells were cultivated in RPMI 1640 mediumsupplemented with 10% FCS (fetal calf serum), Penicillin/Streptomycinand L-glutamine. In subdued light, the medium was removed and mediumcontaining 2 μg/ml TPPS_(2a) was added. The cells (protected from light)were incubated at 37° C. for 18 h. The cells were washed three timeswith medium and for the PLL-L (“light after”) samples 1 ml mediumcontaining a pEGFP-N1/Poly-L-Lysine complex was added. The complexcontained 5 μg pEGFP-N1 and had a charge ratio of poly-L-lysine (PLL) toDNA of 1.7. After 4 h of further incubation at 37° C. in the dark themedium was removed, and the cells were washed once with medium. 1 mlmedium was added and the cells were exposed to blue light as indicatedin FIG. 16 and described under Materials and Methods. For the L-PLL(“light before”) samples the first 4 h incubation was in medium withoutpEGFP-N1/PLL complex, the complex being added immediately afterillumination and removed after a further 4 h incubation. The cells wereincubated for 2 days (still protected from light) prior to analysis forEGFP expression by flow cytometry. For this analysis the cells weretrypsinized (Trypsin-EDTA, Sigma, Mo., USA), resuspended in 400 μl RPMImedium and filtered through a 50 μm mesh nylon filter before analysis ina FACSCalibur flow cytometer (Becton Dickinson, Calif., USA). EGFP wasmeasured through a 510–540 nm filter after excitation at 488 nm.Propidum iodide (1 μg/ml) was used to discriminate dead cells fromviable cells, and pulse-processing was performed to discriminate celldoublets from single cells. 10 000 events were collected for eachsample, and the data were analysed with CELLQuest Software (BectonDickinson, Calif., USA).

Results

As can be seen in FIG. 16 for poly-L-lysine mediated transfection of THXcells the “light before” addition of the transfer molecule approachworks as well as the “light after” approach when using the TPPS_(2a)photosensitiser.

Example 16

Effect of the Light First PCI Strategy on Transfection of THX CellsMediated by poly-L-lysine Using TPPS₄ as the Photosensitiser

THX cells were grown and treated as described under Example 15, exceptthat the TPPS₄ photosensitizer (75 μg/ml) was used instead of TPPS_(2a).

Results

From FIG. 17 it is apparent that for poly-L-lysine mediated transfectionof THX-cells the “light before” approach works slightly better than the“light after” approach when using the TPPS₄ photosensitiser, but bothmethods still achieved transfection.

Example 17

Effect of the Light First PCI Strategy on Transfection of HCT 116 CellsMediated by poly-L-lysine, Using TPPS_(2a) as the Photosensitiser

HCT 116 cells were grown and treated as described under Example 15.

Results

From FIG. 18 it can be seen that for poly-L-lysine mediated transfectionof HCT 116 cells the “light before” approach works as well as the “lightafter” approach when using the TPPS_(2a) photosensitiser.

Example 18

Effect of the Light First PCI Strategy on Transfection of HCT 116 CellsMediated by poly-L-lysine Using TPPS4 as the Photosensitiser

THX cells were grown and treated as described under Example 15, exceptthat the TPPS₄ photosensitizer (75 μg/ml) was used instead of TPPS_(2a).

Results

FIG. 19 shows that for poly-L-lysine mediated transfection of THX cellsthe “light before” approach works as well as the “light after” approachwhen using the TPPS₄ photosensitiser.

Example 19

Effect of the Light First PCI Strategy on Transfection Mediated by DOTAPUsing TPPS₄ as the Photosensitiser

HCT 116 cells were cultivated in RPMI 1640 medium supplemented with 10%FCS (fetal calf serum), Penicillin/Streptomycin and L-glutamine. Insubdued light, the medium was removed and medium containing 75 μg/mlTPPS₄ was added. The cells (protected from light) were incubated at 37°C. for 18 h. The cells were washed three times with medium and for theDOTAP-L (“light after”) samples 1 ml medium containing a complex of 1 μgpEGFP-N1 and 5 μg DOTAP was added. After 4 h of further incubation at37° C. in the dark the medium was removed, and the cells were washedonce with medium. 1 ml medium was added and the cells were exposed toblue light as indicated in FIG. 20 and described under “Materials andMethods”. For the L-DOTAP (“light before”) samples the first 4 hincubation was in medium without pEGFP-N1/DOTAP complex, the complexbeing added immediately after illumination and removed after a further 4h incubation. The cells were incubated for 1 day (still protected fromlight) prior to analysis for EGFP expression by flow cytometry asdescribed under Example 15.

Results

From FIG. 20 it can be observed that for transfection of HCT 116 cellsmediated by the cationic lipid DOTAP the “light before” approach workssubstantially better than the “light after” approach when using theTPPS₄ photosensitiser. The “light before” approach seems to beespecially advantageous for transfection mediated by cationic lipids.

Example 20

Effect of the Light First PCI Strategy on Transfection Mediated bySuperFect® Using TPPS_(2a) as the Photosensitiser

SuperFect® was purchased from QIAGEN AG.

Preparation of Plasmid/Superfect® Complexes

Plasmid/SuperFect® complexes were prepared as follows: (i) pEGFP-N1 wasdiluted with RPMI 1640 medium (without serum, proteins and antibiotics).(ii) SuperFect® (2 μl per μg DNA) was added to the plasmid solution andthe contents were mixed by vortexing for 10 s. (iii) The solution wasincubated for 10–20 min at room temperature to allow complex formation.(iv) 400 μl of cell growth medium (with serum and antibiotics) was addedto the tubes containing the transfection complexes and the contents weremixed by pipetting up and down two times, and the total volume wasimmediately transferred to the cells.

Treatment of the Cells

HCT 116 cells (75000 cells/well, 1 ml/well) were seeded into 12-wellplates (Costar Corning, N.Y., USA) and allowed to attach for six hours.1 ml medium with 0.7 μg/ml TPPS_(2a) was added, and the cells wereincubated for 18 h (5% V/V CO₂, 37° C.). The cells were washed threetimes with medium and incubated for 4 h (37° C., 5% v/v CO₂) inserum-containing medium. The cells were illuminated by exposure to abank of four fluorescent tubes (Osram 18W/67) with the highest fluencearound 420 nm. The plasmid/SuperFect® complexes were added immediatelyafter the light exposure, and the cells were incubated with thecomplexes for 1 or 4 h. The cells were then washed 4 times in RPMImedium, and after addition of 1 ml medium they were incubated furtherfor two days. Then the expression of EGFP was analysed by flow cytometryas described under Example 15.

Results

It can be seen (FIG. 21) that PCI substantially improves SuperFect®transfection under all conditions tested. E.g. for 0.75 μg DNA and 1 htransfection time a 9-fold improvement was seen, while for 0.75 μg DNAand 4 h transfection time a 10-fold enhancement was observed.

Example 21

Effect of the Light First PCI Strategy on Adenovirus Mediated GeneTransduction of HCT 116 Cells Using TPPS_(2a) as the Photosensitiser

HCT 116 cells were cultivated in RPMI 1640 medium supplemented with 10%FCS (fetal calf serum), Penicillin/Streptomycin and L-glutamine. Insubdued light, the medium was removed and medium containing 1 μg/mlTPPS_(2a) was added to each well. The cells (protected from light) wereincubated at 37° C. for 18 h. The cells were washed three times withmedium. The cells were then infected with the Ad-HCMV-LacZ adenovirus atdifferent time points before or after illumination (which was always 4 hafter the removal of the photosensitizer). The cells were incubatedfurther for 2 days (still protected from light) prior to analysis forβ-galactosidase activity by flow cytometry as described under Example 10(Materials and Methods).

Results

FIG. 22 shows the effect of the timing of the light treatment relativeto the delivery of the virus on the PCI effect on adenovirus mediatedgene transduction. It can be seen that for the “light before” approach(the right side of the Y-axis) the PCI illumination is effective for atleast 13 h, so that the virus can be administered up to at least 13 hafter illumination, still maintaining the positive PCI effects ontransduction. This is very important from a clinical point of viewbecause it allows the clinician great flexibility in designing thetreatment and coordinating it to other treatments the patient mightreceive, e.g. to surgical procedures.

Example 22

Effect of the Light First PCI Strategy on Transfection of HCT 116 CellsMediated by Poly-D-lysine Using AlPcS_(2a) as the Photosensitiser

HCT 116 cells were grown, treated and analysed as described underExample 15 except that poly-D-lysine was used instead of poly-L-lysinein making the complex with pEGFP-N1.

Results

From FIG. 23 it can be observed that PCI with the “light before”protocol works well also when the polycation poly-D-lysine is used asthe transfection agent.

Example 23

Effect of the Light First PCI Strategy on Cell Killing by the CytostaticAgent Bleomycin Using TPPS_(2a) as the Photosensitiser

Tetraphenylporphine disulfonate (TPPS_(2a)), lot #04197, was produced byPorphyrin Products (UT, USA). TPPS_(2a) was dissolved in DMSO.

The Chinese hamster lung fibroblast cell line V-79 was used in thisstudy.

MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) wasfrom Sigma (MO, USA; cat. no. M 2128), dissolved in PBS to aconcentration of 5 mg/ml, sterile filtered and stored at 4° C.

Bleomycin (ASTA Medica) 15 000 IE/KY was obtained from the pharmacy atthe Norwegian Radium Hospital. 1 IE corresponds to 1 mg of Bleomycin.The bleomycin powder was dissolved in sterile 0.9% NaCl-solution to afinal concentration of 2 mM.

Cell Cultivation

The V-79 cells were cultured in RPMI 1640 medium (Gibco) supplementedwith 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycinand 2 mM glutamine (all Gibco BRL, Paisley, Scotland) at 37° C. and 5%CO₂ in a humid environment.

Treatment of the Cells

The cells (75 000 cells/well, 1 ml/well) were seeded into 12-well plates(Costar Corning, N.Y., USA) and allowed to attach for 6 h. To some ofthe wells 1 ml medium with 0.7 μg/ml TPPS_(2a) was added (see Table 1),and the cells were incubated for 18 h (5% V/V CO₂, 37° C.). The cellswere washed three times with medium. The cells were then incubated inserum-containing medium for 4 h. The medium was removed, new medium wasadded and the cells were illuminated by exposure to light from a boxcontaining a bank of four fluorescent tubes (Osram 18W/67) with thehighest fluence around 420 nm.

Different doses of bleomycin were immediately added. After 1 or 4 hincubation with bleomycin the cells were washed once with RPMI-medium, 1ml of medium was added and after 3 days of incubation cell survival wasmeasured by the MTT assay. This method is based on reduction of awater-soluble tetrazolium salt (MTT) to a purple, insoluble formazanproduct by mitochondrial dehydrogenases present in living, metabolicallyactive cells. One ml medium containing 0.25 μg MTT is added to thecells, followed by 4 h incubation (37° C., 5% v/v CO₂). The resultingformazan crystals are dissolved by adding 200 μl isopropanol (Sigma,Mo., USA) per well. The solution is transferred to a 96 wells platewhich is read by a Multiskan EX microplate reader (Labsystems, Finland)with a 570 nm bandpass filter. Cell survival is calculated as percent ofcontrol cells not receiving light treatment.

Results

FIG. 24 shows that PCI with the “light before” approach can alsoincrease the biological effect of a low molecular weight, clinicallyapproved chemotherapeutic agent (bleomycin). Thus, it can be seen thatfor the 100 μM bleomycin dose a substantial light-induced increase inthe cytotoxicity of bleomycin can be observed (▪ and ● in FIG. 24). Thelack of effect at the lowest dose of bleomycin (♦ in FIG. 24) shows thatthis increased cytotoxicity is not a result of the photochemicaltreatment per se, since this sample series received the samephotochemical treatment as the 100 μM bleomycin series withoutobservable light-induced effects on cell survival.

Example 24

PCI with Gelonin for Treatment of Tumours in a Mouse in vivo Model

Animals

Balb/c (nu/nu) nude female mice were bred at the Animal Department ofthe Institute for Cancer Research. The mice were kept under specificpathogen-free conditions. Water and poop was given ad libitum. Allprocedures involving mice were carried out in agreement with theprotocols approved by the animal care committee at the Norwegian RadiumHospital, under control by the National Ethical Committee's guidelineson animal welfare. The mice were on average 20–25 g (5–8 weeks old) atthe start of the experiments, and we used at least 5 mice per experimentgroup. The WiDr human adenocarcinoma used in the present study, waspropagated by serial transplantation into the Balb/c (nu/nu) mice. Thetumours were minced to homogeneity by a scalpel and 20 μl of thesolution injected subcutaneously on the right hip of each mouse. Thetumour size was measured two or three times per week by measuring twoperpendicular diameters. Tumour volume was calculated using thefollowing formula:V=(W ² ×L)/2where W is the width and L the length diameters of the tumours measured.Treatment

The mice were randomly allocated to the different groups shown in Table1 and FIG. 25. A stock solution of AlPcS_(2a) was diluted to 1.25 mg/mlin PBS and injected intraperitoneally to a final concentration of 10mg/kg when the tumours had reached a volume of approximately 100 mm³. 48h after the injection of AlPcS_(2a) the tumours were exposed for 16 minto red light (see below). Immediately after light exposure gelonin (50μg total amount in a 2 mg/ml solution, i.e. 25 μl) was injectedintratumorally. The mice were kept in the dark for 1 week after theinjection of AlPcS_(2a).

Light Treatment

The tumours were illuminated with a 150 W halogen lamp (XenophotHLX64640) filtered with a 580 nm long pass and a 700 nm short passfilter emitting 150 mW/cm². The animals were covered with aluminium foilexcept above the tumour area where a hole in the foil with a diameter 2mm larger than the tumour diameter had been made. The tumours wereexposed to 145 J/cm²of light. Tumour volumes were measured two or threetimes per week as described above. Mice were killed when the tumoursreach a diameter of approximately 20 mm. The fraction of tumour-freemice 30 days after illumination was scored (Table 1), and the meantumour volume in each treatment group was recorded (FIG. 25).

Results

TABLE 1 The mice were treated as described above and the occurrence oftumours was recorded 30 days after illumination. FRACTION TUMOUR-FREE %TUMOUR-FREE MICE 30 DAYS MICE 30 DAYS GROUP AFTER AFTER No. TREATMENTILLUMINATION ILLUMINATION 1 Untreated 1/8 13 2 PBS + light 0/7 0 3Gelonin 0/8 0 4 Gelonin + “light 0/5 0 before” 5 AlPcS_(2a)  0/10 0 6AlPcS_(2a) + gelonin 0/7 0 7 AlPcS_(2a) + light  2/11 18 8 AlPcS_(2a) +gelonin + 4/5 80 “light before”

From Table 1 it can be seen that PCI with gelonin using the “lightbefore” approach (group 8) cured 80% (4 of 5) mice from their tumours.In contrast gelonin alone showed no effect, neither with (group 4) norwithout (group 3) additional light treatment (without AlPcS_(2a)).Neither did gelonin in combination with AlPcS_(2a) without lighttreatment (group 6) show any effect. A low cure rate was seen inuntreated animals (group 1) probably due to a spontaneously disappearingtumour. A low cure rate could also be observed for animals receivingAlPcS_(2a) and light treatment (group 7), due to a photodynamic therapy(PDT) effect that was independent on the presence of gelonin. However,this PDT effect (18% cure) was significant lower than what was found forthe PCI treatment with gelonin (80%, group 8). Since gelonin on its ownhad no effect whatsoever the high cure rate in the PCI group cannot beexplained by an additive effect of PDT and gelonin, but must be due to asynergistic effect where the PCI treatment realizes the toxic potentialof gelonin.

FIG. 25 shows the effect of the PCI treatment on the mean tumour volumein some of the treatment groups. It can be seen that in the groupreceiving only gelonin (Δ) the tumours grew as fast as in animals givena placebo treatment of phosphate buffered saline (PBS) injectioncombined with illumination (▪). In animals receiving only thephotochemical treatment, but no gelonin (⋄) the tumour growth wasdelayed, but the tumours started growing again approximately 15 daysafter illumination. In contrast, for the animals receiving the fullgelonin PCI treatment (●) no increase in the mean tumour volume could beobserved even 33 days after illumination.

1. A method for introducing a nucleic acid transfer molecule into thecytosol of a cell, said method comprising contacting said cell with aphotosensitising agent, contacting said cell with said nucleic acidtransfer molecule and irradiating said cell with light of a wavelengtheffective to activate the photosensitising agent, wherein said nucleicacid transfer molecule is taken up by the cell and wherein as aconsequence of said irradiation said nucleic acid transfer molecule isreleased into the cytosol of said cell, wherein said irradiation isperformed prior to the cellular uptake of said nucleic acid transfermolecule into any intracellular compartment.
 2. A method for introducinga nucleic acid transfer molecule into the cytosol of a cell, said methodcomprising contacting said cell with a photosensitising agent,irradiating said cell with light of a wavelength effective to activatethe photosensitising agent and, at the same time or at a time after theirradiation, contacting said cell with said nucleic acid transfermolecule, wherein said nucleic acid transfer molecule is taken up by thecell and wherein as a consequence of said irradiation is released intothe cytosol of the cell.
 3. A method as claimed in claim 1 wherein thecell is contacted with the transfer molecule at a time point afterirradiation has taken place.
 4. A method as claimed in claim 1 whereinthe cell is contacted with said transfer molecule 0 to 4 hours afterirradiation has taken place.
 5. A method as claimed in claim 1 whereinthe cell is contacted with the transfer molecule at the same time as theirradiation.
 6. A method as claimed in claim 1 wherein the transfermolecule is contacted with said cell for 30 minutes to 6 hours.
 7. Amethod as claimed in claim 1 wherein said method is performed on cellsin vitro or in vivo.
 8. A method as claimed in claim 1 wherein said cellis an antigen-presenting cell.
 9. A method as claimed in claim 1 whereinsaid nucleic acid molecule is incorporated into a vector, preferably anadenovirus.
 10. A method as claimed in claim 1 wherein thephotosensitising agent is selected from the group consisting ofmeso-tetraphenyl porphine with 4 sulfonate groups (TPPS₄), tetraphenylporphine with 2 sulfonate groups on adjacent phenyl groups (TPPS_(2a)),aluminum phthalocyanine with 2 sulfonate groups on adjacent phenyl rings(AlPcS_(2a)) and other amphiphilic photosensitizers.
 11. A method asclaimed in claim 1 wherein said photosensitizing agent is a compoundbeing 5-aminolevulinic acid or an ester of 5-aminolevulinic acid or apharmaceutically acceptable salt thereof.
 12. A method as claimed inclaim 1 wherein said photosensitizing agent is contacted with said cellsfor 4 to 24 hours prior to irradiation, preferably for that periodimmediately prior to irradiation.
 13. A method as claimed in claim 1wherein said photosensitizing agent is removed after contact with saidcell for 1 to 4 hours prior to irradiation.
 14. A method as claimed inclaim 1 wherein one or both of the photosensitising agent and thetransfer molecule is affached to, associated with, or conjugated to, oneor more carrier molecules, targeting molecules or vectors.
 15. A methodas claimed in claim 14 wherein the carrier, targeting molecule or vectorto which or with which, the transfer molecule is attached, associated,or conjugated, is an adenovirus, a polycation, a cationic lipid or apeptide or targeted vector.
 16. A method as claimed in claim 15 whereinsaid transfer molecule is attached to, associated with, or conjugated toan adenovirus vector.
 17. A method as claimed in claim 15 wherein saidpolycation is poly-L-lysine or poly-D-lysine.
 18. A method as claimed inclaim 15 wherein said cationic lipid is1,2-bis(oleoyloxy)-3-(trimethylamino)propane (DOTAP).
 19. A method asclaimed in claim 14 wherein said one or more carrier molecules are aliposome or lipid based construct, preferably containing at least onecationic lipid.
 20. A method as claimed in claim 1 wherein the method isperformed in a plurality of cells, and at least 50% of said cells intowhich said transfer molecule is introduced are not killed.
 21. A methodas claimed in claim 1 wherein the irradiation step is 1 to 10 minutes inlength.